Methods and genetic compositions to limit outcrossing and undesired gene flow in crop plants

ABSTRACT

The present invention relates to methods to control the spread of recombinant DNA molecules between sexually compatible plants of differing genetic composition. The invention describes the production of transgenic plants that comprise recombinant traits of interest or concern linked to repressible lethal genes. The lethal genes are blocked by the action of repressor molecules produced by the expression of repressor genes located at a different genetic locus. The lethal phenotype is only expressed after the segregation of the repressible lethal gene construct and the repressor gene following meiosis. The present invention may be employed for both open-pollinated and hybrid seed production systems and may be used to maintain genetic purity by blocking unintended introgression of genes from plants devoid of the specific repressor gene. The invention includes methods that impart traits that are desirable for environmentally responsible heterologous protein production, to genetic material used to impart said traits and to new plants and products derived by said methods.

This is a continuation of U.S. Ser. No. 09/886,208, filed Jun. 22, 2001,now U.S. Pat. No. 6,753,460, which is a continuation of InternationalApplication No. PCT/CA99/01208, having an international filing date ofDec. 22, 1999, and which claims priority of U.S. Provisional ApplicationSer. No. 60/113,549, filed Dec. 22, 1998.

BACKGROUND OF THE INVENTION

The increasing number and diversity of plants containing novel traitsderived from recombinant DNA research present both environmental andcommercial concerns. The concerns arise from the potential for noveltraits to spread by pollen to sexually compatible plants in a natural orcultivated population.

Plants with new and altered traits imparted by genetic technologies andrecombinant DNA technology in particular are now viewed as thecornerstone of the crop biotechnology industry. Currently a considerablenumber of crops plants with novel traits that originated from tissueculture, somatoclonal variation or mutation as well as geneticengineering are undergoing field trials and the first stages ofcommercial release. These plants not only include conventional cropsgrown on an annual basis, but other plants such as trees or shrubs whichcomprise novel traits and are perennial in nature.

Modern crop varieties comprise both individual genes that confer aparticular trait and combination of genes assembled through conventionalplant breeding. Accordingly, as more novel traits are developed andincorporated into modern crop varieties, it is valuable to have a meansto preserve genetic compositions, including those of specific cropvarieties, cultivars or breeding lines. Of particular value is thepreservation of crops which carry traits not usually found in the crop;for example, plants which produce novel oil, meal or other components orthose plants modified to produce speciality chemicals. Additionally,perennial plants such as trees are being produced which carry noveltraits such as altered lignin levels, insect and fungal resistance andherbicide tolerance.

Novel traits are introduced into plants by conventional breeding orgenetic engineering. However, to date neither route provides featuresthat can be routinely used for maintaining germplasm purity, orcontrolling persistence or potential spread of the novel trait. Currentvectors and genetic compositions typically do not address two importantissues: (1) commercial issues such as the prevention of transformed cropplants or elite varieties from contaminating other commercialproductions, or the prevention of introgression of alien germplasm fromclosely related cultivars or plant species, and; (2) environmentalissues such as the removal of transformed crop plants or related speciesthat have acquired the genes in question from non-agriculturalenvironments. Additionally current transformation methods do not providethe means for reducing the introduction of genes via pollen mediatedout-crossing to other cultivars or related species (either wild orcultivated).

The single largest immediate risk for the use of many crops with noveltraits is the risk of contamination among commercial productions of thesame crop species. The risk of a crop species such as oilseed rape orcanola (Brassica napus) to become a weed or to cross with wild weedyrelatives is modest compared with the near certainty of crossing withother commercial productions of canola, especially where largeproduction areas exist. In the past this has not been a significantproblem for farmers and commercial processors for several reasons.First, breeding objectives have been relatively uniform for canola crop;second, only a small number of cultivars have comprised 90-100% of thetotal acreage grown by farmers; and third, the only speciality type,traditionally cultivated, high erucic acid industrial oil cultivars havebeen grown in physical isolation. Accordingly, cross contamination offood quality canola varieties with genes conferring high erucic acid hasnot been a serious issue.

Recently additional unique varieties have been released. These includevarieties that carry recombinant genes which confer tolerance toherbicides and varieties developed by conventional breeding which havevariations in fatty acid profile, such as high oleic acid. Purity ofseed, both during production and harvesting of canola seed for crushingand processing is now a growing issue. Because of the impendingmodification of canola with numerous additional recombinant genes thatimpart different properties to the oil (e.g. high laurate content) orthe use of plants as producers of heterologous proteins such aspharmaceuticals, potentially serious industrial cross contamination maybe anticipated.

These issues extend to many crops in addition to Brassica oilseeds. Inmaize, increasing emphasis on herbicide tolerance, insect resistance anddiversification of modified end products (eg. starch, oil, meal) clearlyindicates that many different traits will be incorporated in the corncrop. As some maize varieties are destined for specialized use, such aswet milling or feed, or even production of pharmacologically importantproteins, the issue of segregation of these speciality types from themainstream is relevant. Considering that corn pollen can sometimestravel significant distances, a genetic means to control pollination isbe highly advantageous.

Similarly, the proximity of perennial plants to their wild relatives isa problem. For instance, a transgenic tree expressing insect tolerancecould cross with a wild species of tree to create a hybrid thatexpresses insect tolerance. Under managed conditions such asplantations, insect resistance would not have a significantenvironmental impact. However, should the insect resistance trait becomewidespread in a natural forest population a serious ecological problemcould result. Insect populations are part of the food chain in a forestsystem and reduced levels of insects could lead to a collapse of thepredator population, which is often native bird species. Accordingly,for unmanaged systems control of the spread of genes that may carryenvironmental consequences is a highly desirable goal.

Currently physical isolation combined with border rows that function aspollen traps have been employed to contain transgenic plants under studyand development. This method, however, is impractical for widespreadcultivation. Moreover, with increasing production and distribution of anincreasing number of different transgenic types, the potential forcontamination increases dramatically. This issue has recently become amajor concern for the oilseed rape industry and will become a greaterissue for other major crops (eg. corn) as the numbers of differentrecombinant and speciality genotypes reach the market place.

In addition to cross-contamination among commercial crop productions,another concern is the potential spread of crops used as vehicles forproducing heterologous proteins of commercial or medicinal value. Thesenovel protein products can potentially contaminate plants destined forfood use and export. Although production standards can be implementedthat will attempt to preserve the identity of individual transgeniclines and reduce unintended contaminations, the outflow of genes toother cultivars will eventually occur. The potential spread of genesthat cannot be easily identified, e.g. by herbicide tolerance, norimpart a distinctive morphology has yet to be addressed by government orindustry.

Methods which control the spread of transgenes into the environment orother commercial cultivars are also useful for preventing theintrogression of alien germplasm into identity-preserved commercialvarieties. In this regard “alien germplasm” is defined as any germplasmwhich does not comprise the full complement of traits of theidentity-preserved cultivar. Accordingly alien germplasm can includeboth sexually compatible wild relatives and other commercial varietiesof the crop. With an increasing number of plants carrying novel traitsbeing contemplated for commercial production, methods that prevent thecontamination of both seed production and commodity production willprovide a valuable means to maintain germplasm purity and identitypreservation.

As an example, many enzymes have been tested that alter plant oilproduction in oilseed crops such as soybean corn and canola. The sameplant species have been used for producing inedible short chain or longchain industrially fatty acids as well as edible oil. Since modified oilseeds must be isolated to ensure pollen carrying the oil modificationgenes does not contaminate edible oil variety seeds, this poses agrowing problem for the seed production industry. The isolationdistances routinely practiced in seed production for many crops may notbe sufficient to ensure required levels of purity. Where crop plants areused to produce speciality products such as pharmaceutically activecompounds, even minor contamination of germplasm is highly undesirable.

Oil seed crops such as canola typically shatter seed before harvest.This results in significant numbers of volunteer plants in subsequentyears, potentially contaminating subsequent commercial productions bothby crossing and by direct effects of the pollen on developing grain(xenia effects). In addition, seeds retained and distributed by farmersfor future planting could contribute to contamination problems.

For perennial plants, the long life of trees and the presence ofindigenous wild relatives raise additional concerns. Some trees takemany years to flower, producing enormous amounts of pollen that can lastfor many years and are especially suited for widespread windpollination. Transgenic trees therefore pose special problems and mayrequire mechanisms to control gene flow to wild relatives.

It has been suggested that some new crop types, through hybridizationwith wild relatives, may invade natural ecosystems. This and relatedissues have been extensively debated (eg. University of California, Riskassessment in agricultural biology: proceedings of an internationalconference, 1990, Casper, R., & Landsman, J., 1992, The bio-safetyresults of field tests of genetically modified plants andmicroorganisms. Proceedings of the 2nd International Symposium on TheBiosafety Results of Field Tests of Genetically Modified Plants andMicroorganisms, 1992 Goslar, Germany, Dale, P. et al., 1992, The fieldrelease of transgenic plants. The British Crop Protection Council.Brighton Crop Protection Conference: Pests and Diseases, Vols. I, II andIII., Proceedings of the 3rd International Symposium on The BioSafetyResults of Field Tests of Genetically Modified Plants andMicroorganisms, 1994, Monterey, Calif., D. D. Jones, 1994).

The consensus of these studies and experimental results achieved to datesupport the view that the degree of potential spread of transgenes towild relatives is highly dependent upon the species and environmentalconditions. Crossing with relatives is not likely with some species andprobable for others (Raybould & Grey, J. Applied Ecology 30: 199-219,1993). Many crops are highly specialized and adapted to non-competitivecultivation practices and thus are not generally considered a seriousenvironmental risk on their own (Dale et al., Plant Breeding 111:1-22,1993, Fishlock, D., The PROSAMO Report, published by the Laboratory ofthe Government Chemist, Queens Road, Teddington, Middlesex, UK TW11 0LY)The potential for environmental problems due to, for example, theinclusion of a virus coat protein gene that has potential for viralrecombination and the evolution of new viruses with an extended hostrange, is currently unknown (Gal S., et al., Virology 187:525-533,Grimsley, N., et al., EMBO Journal 5: 641-646, 1986, Lecoq, H., et al.,Molec. Plant Microbe Interact. 6:403-406, 1993. Tepfer, M.,Biotechnology 11: 1125-1132. 1993). Accordingly there is a need formethods to restrict the potential flow of this type of genes or toselectively eliminate those plants which contain such genes.

Attempts have been made to develop methods to specifically remove oridentify plants that contain novel traits introduced by recombinant DNA.For example, the use of a conditionally lethal gene, i.e. one whichresults in plant cell death under certain conditions, has been suggestedas a means to selectively kill plant cells containing a specificrecombinant DNA. Recently the development of genes which areconditionally lethal in plants have been described (eg WO 94/03619).However, methods using these genes have been restricted to theapplication of a substance that triggers the expression of the lethalphenotype. For widespread agricultural practices, these methods haveserious limitations.

An example of a conditionally lethal gene is the Agrobacterium Tiplasmid-derived oncogene commonly referred to as “gene 2” or “oncogene2”. The gene encodes the enzyme indole acetamide hydrolase (IAMH) thathydrolyzes indole acetamide, a compound that has essentially nophytohormone activity, to form the active auxin phytohormone indoleacetic acid. The enzyme IAMH is capable of hydrolyzing a number ofindole amide substrates including naphthalene acetamide, resulting inthe production of the well known synthetic plant growth regulatornaphthalene acetic acid (NAA). Use of the IAMH gene for roguing plantshas been described by Jorgenson (U.S. Pat. No. 5,180,873). The methodrequires application of NAM to discriminate plants which carry theconditionally lethal gene.

Other enzymes may also be used as conditionally lethal genes. Theseinclude enzymes which act directly to convert a non-toxic substance to atoxin, such as the enzyme methoxinine dehydrogenase, which convertsnon-toxic 2-amino-4-methoxy-butanoic acid (methoxinine) to toxicmethoxyvinyl glycine (Margraff, R., et al., 1980, Experimentia 36: 846),the enzyme rhizobitoxine synthase, which converts non-toxic2-amino-4-methoxy-butanoic acid to toxic2-amino-4-[2-amino-3-hydroxypropyl]-trans-3-butanoic acid(rhizobitoxine) (Owens, L. D, et al., 1973, Weed Science 21: 63-66), thede-acylase enzyme which acts specificlly to convert the inactiveherbicide derivative L-N-acetyl-phosphinothricin to the activephytotoxic agent phosphinothricin (Bartsch, K. and Schultz, A., EP617121), and the enzyme phosphonate monoester hydrolase which canhydrolyze inactive ester derivatives of the herbicide glyphosate to formthe active herbicide (Dotson S. B., and Kishore G. M., 1993, U.S. Pat.No. 5,254,801). Other conditionally lethal genes may be engineered fromlethal genes. A lethal gene which is expressed only in response toenvironmental or physiological conditions is lethal under thoseconditions. For example, a gene that encodes a lethal activity may beplaced under the control of a promoter that is induced in response to aspecific chemical trigger or an artificial or naturally occurringphysiological stress. In this fashion the expression of the lethal geneactivity is conditional on the presence of the inducer.

The expression of the conditionally lethal gene that acts on a non-toxicsubstance to convert said substance to a toxic substance is typicallyregulated by a promoter that is a constitutive promoter expressed in allor most cell types or a developmentally regulated promoter expressed incertain cell types or at certain stages of development. Any promoterthat provides sufficient level of expression can be used. However, inpractice promoters that provide high levels of expression for extendedperiods offer the best opportunities to remove unwanted plants.

The need to apply a chemical to induce the lethal phenotype reduces theutility of a conditionally lethal gene. The widespread application ofchemicals may be impractical and raise additional environmentalconcerns. Accordingly the use of conditionally lethal genes as currentlydescribed is not ideally suited for general applications sinceintervention is required to express the lethal phenotype.

The possibility of using a repressed lethal gene to limit thepersistence of hybrid crops has been suggested recently by Oliver et al(patent application WO 96/04393). In this system expression of a lethalgene is blocked by a genetic element that binds a specific repressorprotein. The repressor protein is the product of a repressor genetypically of bacterial origin. The genetic element that binds therepressor protein is referred to as a blocking sequence and isconstructed such that it further comprises DNA sequences recognized by aDNA recombinase enzyme (e.g. the CRE enzyme). Plants that contain saidblocked lethal gene are hybridized with plants comprising the DNArecombinase gene. Either the lethal gene or the recombinase enzyme (orboth) is under control of regulatory elements that allow expression onlyat a specific stage of plant development (e.g. seed embryo).Consequently, the recombinase function in the resulting F1 hybrid plantremoves the specific blocking sequence and activates the lethal gene sothat no F2 plant is produced. Notably, this scheme cannot controloutcrossing of germplasm that carries the novel trait nor introgressionof alien germplasm. The method does not apply to self- oropen-pollinating varieties. Accordingly, the method is useful only as ameans to restrict use (e.g. re-planting) to F1 hybrid seed.

Methods to eliminate recombinant DNA sequences used to obtaintransformants such as selectable markers have been developed. Use of atransposase or recombinase to remove selected recombinant sequences fromtransgenic crop plants has been described in U.S. Pat. No. 5,482,852(Biologically Safe Transformation System, by Yoder and Lassner). Thisinvention describes a method for removing vector and marker genesequences by enclosing them within a transposon. The sequences aresubsequently removed by crossing the plant to a plant with transposasefunction.

No published method, however, addresses the problem of contamination ofrelated varieties by cross pollination. The art also does not provide ameans to prevent the introgression of alien germplasm by pollinationwith related pollen, even pollen from the same variety but lacking thegenetic trait(s).

Therefore, a method that limits outcrossing and introgression withoutintervention is needed for management and control of novel traits andcrops with novel traits. A mechanism to control cross-contaminationsamong commercial crops is also needed. Such a mechanism is also neededin the management of perennial crops such as trees, shrubs andgrapevines. In particular any mechanism which does not requireintervention in order to function is ideally suited for perennial crops.The present invention describes methods and genetic compositions whichrespond to these needs.

SUMMARY OF THE INVENTION

The present invention comprises methods and recombinant DNA compositionsthat block the spread and persistence of genes in other cultivars of thesame species or related species, resulting from unintended outcrossingby pollen produced by plants containing said recombinant DNA. Theinvention further ensures that introgression of alien germplasm iseliminated in a selfing population.

The present invention relates to novel recombinant DNA constructs thatimpart a novel feature to plants containing the recombinant DNA. Thisfeature permits viable seed to be formed only on plants that contain thefull complement of the recombinant DNA. The present invention furtherprovides a means to ensure the sexual isolation of germplasm or genetictraits within a defined population through the expression of a traitthat is lethal in plants which do not comprise the full complement ofthe recombinant DNA. The invention ensures that those plants which arefertilized by the transgenic plant but which do not carry therecombinant DNA are unable to form viable seed.

The novel genetic constructs impart no morphologically obvious or easilydetectable phenotype to plants. They comprise silent genes that areexpressed only when an unintended sexual cross occurs. An unintendedcross results in expression of a lethal trait and the undesired plantcells are eliminated. Accordingly the invention restricts the formationof viable seed via outcrossing with sexually compatible species. Thenovel DNA constructs further provide a means to effectively reduce theintrogression of traits from cross-pollination with pollen from sexuallycompatible species that lack the constructs.

The present invention provides a genetic trait encoded within DNAconstructs that ensures that specific cultivars or breeding lines arenot contaminated with alien germplasm or contaminate other cultivars andbreeding lines. This provides a convenient means to genetically isolatethe transgenic plant. The novel DNA constructs may be used as a means toensure germplasm purity during seed production and the production of thecommodity in the field and can be used in both open pollinated andhybrid crop varieties.

Linkage of the novel DNA constructs to DNA molecules that encode novelagronomic or phenotypic traits ensures that the novel agronomic orphenotypic trait does not persist outside of the genotype into which itwas introduced. This aspect of the invention is useful in the managementof crops with novel agronomic or phenotypic traits or crops with uniquecombinations of conventional traits developed through plant breedingtechniques.

In one embodiment, the invention provides a genetic system comprisingtwo DNA constructs. One DNA construct comprises a dominant repressiblelethal gene that, when active, results in cellular death, and whoseexpression is inhibited in plant cells which contain a second DNAconstruct comprising a repressor gene, the repressor gene being locatedat a locus that segregates independently from the repressible dominantlethal gene. The repressor gene encodes a repressor molecule which maybe a DNA binding protein, a direct inhibitor of the lethal geneactivity, or an RNA, ribozyme or antisense RNA capable of inhibiting thelethal phenotype.

FIG. 1 illustrates the genetic constructs that may be employed in thisembodiment of the invention

In a preferred embodiment, the dominant repressible lethal gene is underthe control of a seed specific promoter and the gene encoding arepressor molecule is located at a locus that segregates independentlyfrom the repressible dominant lethal gene. Both the repressible dominantlethal gene and the repressor gene are in the homozygous state. Selfpollination maintains this genetic combination.

In another preferred embodiment, the DNA construct further comprises aconditionally lethal gene linked to the repressible lethal gene. Theconditionally lethal gene can be activated by the application of achemical or physiological stress, ensuring a means to completelyeliminate the plants or cells containing the recombinant DNA from theenvironment when required. Accordingly, even self-pollinated cellscontaining a repressible lethal gene can be selectively removed from apopulation by virtue of the conditionally lethal gene.

In an additional preferred embodiment, the repressible lethal genelinked to a conditionally lethal gene is linked additionally to a geneencoding a novel trait. A second DNA construct comprises a gene encodinga repressor capable of blocking the activity of the repressible lethalgene. The separate DNA constructs are introduced into the same cells.Linkage of the novel trait to the repressible lethal gene ensures thatthe novel trait can not persist in related species by transfer throughsexual crossing.

In a still further embodiment, the DNA constructs comprising therepressible lethal gene and the repressor gene are within a singlerecombinant DNA molecule which is introduced into the plant cell. Thesingle recombinant DNA molecule further contains sequences recognized bya site specific recombinase or transposase. Recombinase or transposaseactivity results in the removal of the repressor gene from the insertedrecombinant DNA. As an element of this embodiment, the repressor gene isreintegrated to an independently segregating locus; in particular, tothe same locus on the opposite chromosome of a homologous chromosomepair. The DNA constructs that may be employed within the scope of thisembodiment are illustrated in FIG. 2.

In another preferred embodiment, DNA constructs are introduced into aplant cell, comprising two repressible lethal genes and two functionallydistinct repressors for the repressible lethal genes. The genes arepreferably arranged so that the first repressible lethal gene is linkedto the repressor capable of repressing the second repressible lethalgene, and the second repressible lethal gene is linked to the repressorcapable of repressing the first repressible gene, as illustrated in FIG.3. Optionally, the constructs comprise a single recombinant DNA moleculewhich is introduced into the plant cell. The single recombinant DNAmolecule contains sequences recognized by a site specific recombinase ortransposase, whose activity results in the removal of the firstrepressible lethal gene and the second repressor from the recombinantDNA. As an element of this optional embodiment, plants are selectedwherein the first repressible lethal gene and the linked secondrepressor gene are reintegrated to an independently segregating locus.

The foregoing embodiments rely on random insertion of the DNA constructsto loci that segregate independently. However, for some applications ameans to introduce the recombinant DNA to a specific locus may bedesirable. Accordingly, the present invention provides methods to targetthe recombinant DNA to a specific locus.

The use of a site specific recombinase to introduce recombinant DNA to alocus previously established in the plant genome is contemplated. Arecombinase target DNA sequence recognized by a site specificrecombinase is inserted into the plant genome by standard transformationprocedures. The plant is made homozygous for the target DNA sequence byknown methods such as selfing and selection or anther or isolatedmicrospore culture. Alternatively a plant homozygous for said insertedsequence can be made directly by transformation of haploid cells ortissue, followed by chromosome doubling.

The appropriate recombinase expressible in plant is inserted by any ofseveral methods such as transformation, microinjection,electroportation, etc. into plant cells homozygous for the target DNAsequence. The plant cells are then independently re-transformed with DNAconstructs comprising either the repressible lethal gene or therepressor gene. These DNA constructs have been modified to include sitespecific recombinase recognition sequences such that the DNA constructcan be inserted into the pre-existing recombinase target DNA sequence.Accordingly, plant lines are recovered that contain either the DNAconstruct comprising the first repressible lethal gene or the firstrepressor gene. By crossing said lines, plants may be recovered thatcontain both introduced DNA constructs (repressible lethal gene andrepressor) at the same genetic locus on opposite chromosomes of ahomologous chromosome pair.

Accordingly the site-specific insertion method comprises preparation ofDNA constructs comprising a repressible lethal gene and in someembodiments a dominant conditionally lethal gene. The method alsocomprises preparation of a repressor gene which can be insertedconcomitantly or independently of the lethal gene. The repressiblelethal gene is repressed by the repressor encoded by the repressor gene,conveniently located at a chromosomal site that segregates independentlyof the inserted repressible lethal gene. It is within the scope of thepresent method to employ site-specific recombination as a means totarget repressor and repressible lethal genes to specific sites withinthe plant genome, in particular to those sites at which specificrecombinase recognition sites have been inserted. An illustration of theDNA constructs and steps that may be employed in this embodiment of theinvention are shown in FIG. 4.

The invention provides methods and compositions that allow the geneticpurity of transgenic plants to be maintained by simple self pollinationin open pollinated crops. No intervention is required. The inventionfurther provides methods for the convenient preparation, propagation andhusbandry of plants containing the recombinant DNA. Genetic compositionsare provided for use in open pollinated and hybrid plant productionsystems. Illustration of the utility of the method as employed with openpollinated crops such as Brassica napus oilseed is shown in FIG. 5,illustration of the utility of the method as employed with hybrid cropssuch as maize is shown in FIG. 6.

During the production of pollen, the repressible lethal gene issegregated from the repressor gene, in accordance with the geneticschemes described above. Subsequently any out-crossed plants (i.e. thoseplants that have inadvertently received pollen that carries therepressible lethal gene) cannot form viable seed because the newlyformed seed contains no repressor to repress expression of the lethalgene. The lethal gene is repressed in selfed plants because these plantsretain both lethal and repressor genes. For those embodiments whichfurther comprise a conditionally lethal gene linked to the repressiblelethal gene, plants containing these genes can be eliminated byapplication of a chemical or physiological stress to activate theconditionally lethal gene.

The present invention provides methods and compositions for theproduction of recombinant plants with substantially reduced or zero riskof gene transfer via crossing. In some embodiments, the plants can besafely and specifically removed from the growing site by application ofan inexpensive and environmentally benign chemical.

The invention is well suited to the production of crop plants for largescale agricultural and industrial applications where the potentialcontamination of other commercial productions of the same species, viacross pollination or volunteer seed, is to be avoided. The inventionfurther provides a mechanism of safe use and environmental protectionfor recombinant plants that may cause environmental damage by invasionof other habitats or that may spread their transgenes by crossing bycrossing with wild weedy relatives.

The present invention provides specifically a method for producing cropplants as heterologous protein producers, without risk of contaminatingother commercial productions of the same species.

The invention further provides a means to control the introgression ofalien germplasm into commercial plant varieties and to maintain geneticpurity of lines comprising the introduced genes. It is noted that theDNA constructs comprising these genes can be used with or without beinglinked to a novel trait gene to provide a means of ensuring geneticpurity during seed production or production of the commodity.

For some crops, such as self-incompatible crops, the invention improveshybrid seed production via self-incompatibility. In this particularembodiment of the invention, a self-incompatible female parent ismodified to carry the repressible lethal gene but not the repressorgene. The female line is unable to form viable seed. Crossing thisself-incompatible female parent with pollen that carries a repressorgene results in the production of viable hybrid seed that carries boththe repressible lethal gene and the repressor gene. Linkage of a noveltrait such as insect resistance to the repressible lethal gene wouldfurther prevent the dissemination and persistence of the trait inrelated species.

The use of repressible lethal genes in self-incompatible cropseliminates the problems of breakdown of self-incompatibility in thefemale parent often seen in commercial seed production. This breakdownproblem leads to self-seed contamination of the hybrid seed. By usingrepressible lethal genes, self-seed is not possible on the female parentsince it lacks the repressor and is self-incompatible. A convenientmeans to maintain the female line (such as use of a repressor inducibleunder certain conditions) can be employed to increase the number offemale parents. Alternatively, the line can be clonally propagated.Current mechanisms to overcome self-incompatibility include elevatedcarbon dioxide and other stress treatments. It is within the scope ofthe invention to use promoters that are inducible under the sameconditions as those used to overcome self-incompatibility, as thisprovides a particularly convenient means to increase seed production ofthe female parent. The method is particularly useful for production ofBrassica vegetable crops where self incompatibility is commonly applied.

The following terms are defined and used within the scope of thisinvention.

-   Alien germplasm: a gene or combination of genes or genetic traits    which is not part of the specific genetic makeup of an individual    crop plant or variety.-   Blocking or “blocks”: the inhibition of a lethal gene activity by a    repressor; blocking can include: the prevention of RNA transcription    by binding of a repressor to a specific DNA sequence, binding of an    antisense RNA or ribozyme to a primary RNA or mRNA transcript,    binding of an inhibiting factor to a lethal gene product such as a    RNAse or protease inhibitor binding to a toxic ribonuclease or toxic    protease. Any method which prevents the expression of a lethal    phenotype can be considered as “blocking” the lethal phenotype.-   Conditionally lethal gene: a gene which confers on a plant cell a    phenotype which renders the plant cell sensitive to an agent, said    agent may be genetic or chemical in nature, said sensitivity    ultimately leading the death of the plant cell.-   Constitutive promoter: a DNA sequence capable of causing gene    expression in substantially all plant cells, tissues and organs.-   De-repressed lethal gene: a lethal gene that expresses the lethal    phenotype due to the absence of a functional repressor.-   Gene: a DNA expression cassette comprising a transcribed region    under the control of a promoter further comprising a transcription    termination signal.-   Inducible promoter: a DNA sequence capable of causing gene    expression in response to a chemical, physical or environmental    inducer.-   Introgression: the undesired movement of a gene or genes through    sexual crossing, usually by pollen, from a plant which is not    intended to be the pollen donor for the formation of seed.-   Lethal gene: a gene, that when expressed in a plant cell ultimately    leads to the death of the plant cell.-   Lethal gene activity: a genetic activity that leads to plant cell    death. A lethal gene activity can be due to a single gene or can    also be the result of the combined expression of more than one gene.-   Oncogene: a gene encoding an enzyme involved in tumor formation or    abnormal plant growth as a result of infection of susceptible plants    by Agrobacterium sp. Known oncogenes include those comprising the    tmr and tms loci of the T-DNA region of the Ti plasmid.-   Outcrossing: the movement of pollen from a plant of one genetic type    to a sexually compatible plant of a different genetic type.    Outcrossing is generally used to describe the unintended movement of    pollen; however in some plant species, particularly those which are    self-incompatible, outcrossing is also used to represent the normal    pollination events within a population of incompatible plants.-   Promoter: a DNA sequence capable of causing gene expression in a    plant cell.-   Repressor: a gene product that can specifically block the activity    of a gene product or expression of a gene. A repressor can be a    protein, RNA or a specific substance produced by the activity of a    repressor gene product.-   Repressor binding site: a DNA sequence that is recognized    specifically by a repressor, said recognition leading to the    inhibition of expression of a gene containing said repressor binding    site. In some embodiments a repressor binding site may also    represent a RNA sequence which is recognized by a ribozyme or    antisense RNA.-   Repressor gene: a DNA expression cassette capable of expressing a    functional repressor.-   Repressed lethal gene: a lethal gene where the lethal phenotype that    is a result of the gene activity is blocked by the presence of a    repressor.-   Repressible lethal gene: a lethal gene, the expression of which can    be inhibited by the action of a specific repressor molecule.-   Responsive to a repressor: a lethal gene or lethal gene product the    lethal activity of which is inhibited in response to the presence of    a repressor of lethal gene activity.-   Selfing: self-pollination leading to the formation of a seed or    reproductive structure.-   Tissue specific promoter: a DNA sequence capable of causing    substantial gene expression only in a specific plant cell, organ or    tissue.-   Transcribed region: a DNA sequence that is transcribed under the    control of a promoter. Said DNA sequence may encode a RNA capable of    being translated into a protein or may encode a RNA that can    specifically inhibit or prevent the expression of a gene.-   Transcription termination sequence: a DNA sequence that defines the    termination of transcription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a scheme wherein a repressible lethal gene and arepressor gene are located at independently segregating loci. The term“new trait” represents a linked recombinant DNA or a specific genotypewhich comprises a combination of one or more traits. PRO 1 representsthe promoter controlling expression of the repressible lethal gene; aseed specific promoter is preferred. Sufficient expression of therepressor gene prevents expression of the lethal phenotype.

FIG. 2 illustrates a DNA construct comprising both the lethal gene andthe repressor gene. The repressor gene can be specifically targeted to anew chromosomal location by the use of a site specific recombinase ortransposase. The transposase or recombinase recognition sequences allowthe repressor gene to be re-located, in the presence of activerecombinase or transposase enzyme, to a locus which segregatesindependently of the repressible lethal gene.

FIG. 3 illustrates DNA constructs comprising two repressible lethalgenes and two independent repressor genes. The new trait or traits canbe linked to one or both or the repressible lethal genes. The repressorgenes are functionally distinct, i.e. they act independently. Therepressible lethal genes may encode the same or different repressiblelethal gene activity. PRO 1 and PRO 2 may be the same or differentpromoters; seed specific promoters are preferred.

FIG. 4 illustrates the scheme for producing plants containing arepressible lethal gene where the repressor and lethal genes are locatedon opposite chromosomes of a homologous chromosome pair. A site-specificrecombinase targets the repressor and lethal gene constructs to oppositesister chromosomes of a homologous chromosome pair. The “TargetRecombinase Sequence” may further comprise an inactive selectable markerwhich is activated upon insertion of the construct.

According to this scheme, an elite parental line is transformed with atarget recombinase sequence. Hemizygous plants are recovered andconverted to homozygous state. The plants are re-transformed with arepressible lethal gene or a repressor gene flanked by recombinasetarget sequence(s). Plants are recovered that comprise randomlyintegrated SL or R. Recombinase function is then used to specificallyexcise and insert SL or R into the target recombinase sequence presenton the target chromosome pair. Plants are recovered which contain SL andR on opposite sister chromosomes of a chromosome pair.

FIG. 5 illustrates the use of a repressible lethal gene in selfpollinating crops. Use of a conditionally lethal gene is optional.

In this figure, Recombinant DNA 1 is Repressible Seed Lethal (SL) andConditional Lethal in Other Tissues (CL). Recombinant DNA 2 is aRepressor.

FIG. 6 illustrates the use of a repressible lethal gene method in hybridcrops. Use of a conditionally lethal gene is optional.

In this figure, Recombinant DNA 1 is Repressible Seed Lethal (SL),Conditional Lethal in Other Tissues (CL), also including an InducibleRepressor (IndR). Recombinant DNA 2 is Non-Lethal Repressor (R).

FIGS. 7A and 7B illustrate the isolation of the lethal genes oncogenes 2(FIG. 7 a) and 1 (FIG. 7 b) from the Ti plasmid pTi15955 of theAgrobacterium tumifaciens strain ATCC 15955.

FIG. 8 illustrates the construction of the seed specific promoter, thephaseolin promoter, modified to contain a bacterial repressor bindingsite. The vector containing this modified promoter is pPHAStet1.

FIGS. 9A and 9B illustrate the construction of a plant transformationvector comprising: 1) the oncogene 1 of Agrobacterium lumifaciens Tiplasmid pTi15955 under the control of the phaseolin promoter modified tocontain a bacterial repressor binding site (FIG. 9 a) and, (2) theconditionally lethal gene, oncogene 2, under the control of its nativepromoter (FIG. 9 b).

FIG. 10 shows the germination of wild-type (WT) seeds compared to seedscontaining a repressible seed lethal gene without a repressor gene (theseed lethal or SL phenotype).

FIG. 11 shows wild-type plantlets, as well as plantlets of a segregatingpopulation of plants containing a repressible seed lethal genegerminated under selective and non-selective conditions.

FIG. 12 provides a comparison of plants containing a repressible seedlethal gene (A); plants containing a repressor gene (B); plantscontaining a repressible seed lethal gene and a repressor gene (C); andwild-type plants (D).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the subject invention, methods and compositions areprovided for a novel means of producing transgenic plants wherein thetransfer and persistence of recombinant genes via pollen from saidplants to other cultivars or related species is substantially reduced.Additionally, the methods permit the production of self pollinatingplant lines which carry DNA constructs that restrict outcrossing of thegermplasm and, furthermore, restrict the introgression of aliengermplasm even in sexually compatible plant species.

Accordingly, the method provides genetic isolation and identitypreservation of the germplasm which contains recombinant moleculescomprising repressible lethal genes and repressor genes. The methodfurther allows a means to remove recombinant plants from any growinglocation by application of a chemical agent or exposure of the plants toa physiological stress.

In a first embodiment, the invention provides a method comprising:

I.) Preparing a first DNA expression cassette that comprises, inaddition to the DNA sequences required for transformation and selectionin plant cells, a DNA sequence that encodes a product that is harmful ordisruptive to cells such that death of cells and ultimately death of theentire plant occurs (a lethal gene). Expression of the lethal gene isregulated by an appropriate promoter, preferably a seed specificpromoter. Said lethal gene construct additionally comprises DNA elementsresponsive to a repressor wherein expression of the lethal gene activityis blocked in the presence of said repressor molecule. A gene encoding atrait of interest (novel trait) can be linked to the lethal gene;

II.) Preparing a second DNA expression cassette that comprises, inaddition to the DNA sequences required for transformation and selectionin plant cells, a repressor gene that encodes a repressor moleculecapable of blocking the expression of the lethal gene activity containedin the first DNA expression cassette. Expression of the repressor geneis regulated by a promoter active in plant cells, preferably a promoterthat is expressed in all plant cells, more preferably a promoter that isexpressed at a level and time sufficient to inhibit the expression ofthe lethal gene, and,

III.) Inserting the recombinant DNA described in (I) and (II) into aplant cell capable of being transformed, regenerating the cell into awhole plant, and recovering a plant which contains the DNA of (I) and(II) at positions in the plant genome wherein the DNA of (I) and (II)assorts independently during meiosis.

The gene encoding a “novel trait” can be any recombinant protein orpeptide of interest. Typically this “novel trait” is a heterologousprotein of commercial interest or a protein that confers anagronomically useful trait such as herbicide tolerance. Transfer of thenovel trait through crossing with native or cultivated sexuallycompatible plants which lack the repressor is limited because the lethalphenotype appears in the seed, leading to abortion of seeds which havereceived the novel trait gene. It is further contemplated that thelethal gene activity can comprise a single encoded product or two ormore independent gene products that act cooperatively to express thelethal phenotype.

To maximize independent assortment, commonly referred to as“segregation”, of the repressible lethal DNA and repressor constructsduring meiosis, the recombinant DNA molecules are located on differentchromosomes. Standard methods of transformation are known to result inrandom insertion of recombinant DNA within the plant genome; thus it isexpected that in the majority of plants the recombinant DNAs will belocated on different chromosomes. Independent assortment of genes andthe location of inserted DNA are determined by simple well knownmethods.

Seed increases of plants with the repressible lethal and repressorconstructs located on different chromosomes can be made by simpleselfing in isolation, provided that the plants are homozygous for thetwo recombinant DNA constructs. Such homozygous plants can be obtainedby selfing primary transformants, or by anther or microspore culture ifthe transformation procedure is carried out with diploid tissues.Alternatively, such plants can be obtained directly via transformationof microspores or other haploid cells followed by chromosome doubling.It is apparent to those skilled in the art that plant lines homozygousfor both the repressible lethal gene and the repressor can be obtainedby crossing isogenic transformed plant lines comprising either therepressible lethal gene or the repressor. Alternatively a combination ofsimple tissue culture techniques such as anther culture and sexualcrossing can be employed to recover plants homozygous for both insertedDNAs.

Said homozygous plants described above may be grown on a commercialscale as an open-pollinated crop. The out-crossing of such plants tonon-recombinant sexually compatible plants produces a first generationof plants heterozygous for the recombinant traits. In subsequentgenerations of out-crossed plants the independent segregation of genesduring meiosis will result in a rapid decline in the incidence of plantsexpressing the novel trait. Additionally a variation of the method iscontemplated such that the repressible lethal-novel trait constructfurther comprises a conditionally lethal gene. Plants comprising such agene construct may be removed if required by activating the lethalphenotype, e.g. by chemical spray.

The homozygous plants described above may be grown on a commercial scaleas a hybrid crop if such plants also comprise a pollination controlsystem that allows hybrid seed formation. Such a pollination controlsystem could be any of the known types of male sterility systems such ascytoplasmic male sterility, self-incompatibility or genic malesterility. Additionally male sterility may in some species be achievedby mechanical means or may result from the application of chemicals thatspecifically kill pollen (gametocides). The choice of the appropriatesystem will vary with the individual crop species. Such methods are wellknown to those skilled in plant breeding. The use of plants homozygousfor the repressible-lethal and repressor recombinant gene constructs aseither a male or female parent in a hybrid cross will result in hybridseed heterozygous for the recombinant DNA constructs. Segregation ofthese constructs in the F2 and subsequent generations will result in therapid loss of plants comprising the introduced novel recombinant trait.

Random insertion of recombinant DNA will on some occasions result in theincorporation of said recombinant DNAs on opposite chromosomes of ahomologous chromosome pair. The frequency of occurrence of such eventsis dependent on the number of chromosome pairs comprising the geneticconstitution of a given plant and will occur with greatest frequency inplants with small numbers of chromosome pairs. The introduction of therecombinant DNA constructs to different members of a homologouschromosome pair has the advantage that during meiosis, segregation ofthe repressible lethal-novel and repressor constructs occurs immediatelyand completely such that, provided that recombination due to crossingover has not occurred, no plants containing both constructs are formedas a result of out-crossing. This particular variation of the inventionis particularly suited to development of recombinant crops wherein thetrait of interest, (e.g. production of hormones or otherpharmaceutically active molecules) needs to be very tightly controlled.

Seed increases of plants wherein the recombinant DNA constructs haveinserted into different members of a homologous chromosome pair requirethat the plant cells are essentially heterozygous with respect to therepressible lethal gene and the repressor, to ensure repression of thelethal phenotype. This can easily be achieved by linking the repressiblelethal gene to a selectable marker gene that confers resistance to aspecific chemical. The use of herbicide resistance genes for themaintenance and selection of plants carrying specific recombinant traitsis well documented in the literature and can be employed in the presentinvention. Any gene that confers field level resistance can be used. Itis also possible for a chosen gene, such as the phosphothricin acetyltransferase (pat) gene conferring tolerance to phosphinothricin, to beused for selection during transformation. Plants comprising said geneticconstructs are selfed and seed grown out under field conditions andsprayed with herbicide. Plants homozygous for the repressible-lethalnovel trait (25%) will be killed by the action of the lethal gene in theabsence of the repressor. Plants homozygous for the repressor gene (25%)will be killed by the action of the herbicide. In contrast, plantscontaining both the repressible lethal-novel gent and the repressor gene(50%) will be unaffected. Linkage of a novel trait gene to therepressible lethal gene ensures that the novel trait gene can not formviable seed by inadvertent transfer of pollen to any unintended sexuallycompatible species. Persistence of the novel trait in unintended plantpopulations is therefore completely restricted.

For some applications, control of spread of the novel trait is optimalif two repressible lethal genes and repressor genetic constructs areemployed.

According to this aspect of the invention, methods and compositions areprovided for a novel means of producing transgenic plants that containtwo recombinant repressible lethal gene constructs. All plantscomprising recombinant DNA resulting from outcrossing of the transgenicplant are rapidly eliminated from the environment. The first repressiblelethal gene construct comprises a lethal gene and a repressor gene thatblocks the expression of a second repressible lethal gene and optionallya gene encoding a novel trait of interest. The second repressible lethalgene construct comprises a second lethal gene and a repressor gene thatblocks the expression of the first repressible lethal gene. Cellscontaining both genetic constructs produce two types of repressormolecules; hence both lethal genes remain in a repressed state.Segregation of the genetic constructs during meiosis results inseparation of repressor and lethal genes resulting in ultimate death ofall plants containing any recombinant DNA from the plant whichoriginally contained the two repressed lethal genes.

Thus, in accordance with another aspect of the invention, methods areprovided for a novel means of producing transgenic plants, comprising:

I.) Preparing a first DNA expression cassette that comprises, inaddition to the DNA sequences required for transformation and selectionin plant cells, a first lethal gene. Expression of the first lethal geneis regulated by an appropriate promoter, preferably a seed specificpromoter. This first lethal gene expression cassette contains a firstrepressor responsive site, allowing expression of the lethal gene to beinhibited by a first repressor molecule. Optionally, linked to the firstDNA expression cassette is a third DNA expression cassette comprising adominant conditionally lethal gene and a fourth DNA expression cassettecomprising a second repressor gene encoding a second repressor that isfunctionally distinct from the first repressor molecule and is capableof inhibiting expression of a second lethal gene. A gene encoding anovel trait may also be included;

II.) Preparing a second DNA expression cassette that comprises, inaddition to the DNA sequences required for transformation and selectionin plant cells, a first repressor gene that encodes a first repressormolecule capable of inhibiting the expression of the lethal genecontained in the first DNA expression cassette. Linked to the second DNAexpression cassette is a fifth DNA expression cassette comprising asecond repressible lethal gene, the expression of which is repressed bythe second repressor molecule contained in fourth DNA cassette; and

III.) Inserting the recombinant DNA described in (I) and (II) into aplant cell capable of being transformed, regenerating the cell into awhole plant, and recovering a plant which contains the DNA of (I) and(II) at positions in the plant genome wherein the DNA of (I) and (II)segregates during meiosis and outcrossing.

The first, third or fourth DNA cassette can be linked to a gene encodinga novel trait such as but not limited to, a recombinant protein orpeptide of commercial or agronomic interest. The resultant transgenicplants would carry a trait whose capacity to persist in native orcultivated sexually compatible plants is substantially diminished. Thisis because the latter plants lack the repressor. Any seed resulting fromthe union would therefore express the lethal gene and be aborted.Accordingly, persistence of either recombinant DNA of (I) or (II) in anunintended genotype (i.e. not comprising the complete recombinant DNAcomplement) is inhibited. Plants which contain the first (I) recombinantDNA may also be discriminated by the use of the conditionally lethalmarker gene.

It is further contemplated that the lethal gene activity can comprise asingle encoded product or two independent gene products that actcooperatively to express the lethal phenotype. It is further noted thatthe conditionally lethal gene may comprise a product that can act inboth in cooperation with said repressed lethal gene to express thelethal phenotype or in response to exogenously applied substances thatcan be acted upon directly to cause the expression of the lethalphenotype.

The foregoing embodiments rely on the random insertion of DNA during thetransformation process to achieve the placement of the two recombinantDNAs at loci which segregate during meiosis. This is achieved as aresult of simple crossing and progeny analysis, or by mapping of theinserted DNA using any techniques widely practiced by plant breeders.Accordingly a desired genetic combination is obtained.

However, it is within the scope of the present invention to introducesimultaneously all the required DNA expression cassettes within a singlemolecule, and then use a transposase to transpose the desiredcassette(s) Specific transposition can occur by providing theappropriate combinations and orientations of DNA sequences recognized bya recombination-enzyme such as a transposase.

Known transposons and associated transposase activities include Ac/Dsand En/Spm elements from maize (e.g. see Federoff, N. Maize TransposableElements. In Berg, D. E. and Howe, M. M. (eds) Mobile DNA, pp. 375-411,American Society for Microbiology, Washington, D.C., 1989), Tam-1 andTam-3 from snapdragon (e.g. see Sommer et al, Transposable Elements ofAntirrhinum majus. In Plant Transposable Elements, O. Nelson, ed, PlenumPress, New York, pp. 227-235, 1988), Tnt-1 from tobacco (Pouteau, S. etal, Mol Gen Genet. 228:233-239, 1991), Tph-1 from petunia (Gerats A. G.M. et al, The Plant Cell. 2:1121-1128, 1991) and the Tst-1 element frompotato (Koster-Topfer, et al, Plant Mol. Biol. 14:239-247,1990).

Some transposons may have transposition characteristics that are ofparticular use in the present invention. For example, Ds elements have atendency to transpose over relatively short distances on the samechromosome (Dooner and Belachew, Genetics 122:447-457, 1989, Dooner etal, The Plant Cell 3:473-482, 1991, Jones et al, The Plant Cell2:701-707, 1990, Osborne et al, Genetics 129:833-844, 1991, Rommens etal, Plant Molecular Biology, 20: 61-70, 1992). Such a transpositionpattern would facilitate recovery of a genetic combination where atransposed repressor is transposed to a site on the opposite chromosomeof a chromosome pair that carries the repressible lethal gene.

Accordingly, use of a specific transposase enzyme to move a repressorgene to a genetic locus that segregates independently from therepressible lethal gene is provided as follows:

A DNA construct is modified to contain, in addition to a firstrepressible lethal gene, a repressor gene linked to the firstrepressible lethal gene. The repressor gene is further modified byhaving at its 3′ and 5′ ends specific DNA sequences recognizable by atransposase enzyme. The recognition sequences are oriented in such afashion as to permit the excision of the repressor gene by atransposase. The transposase further catalyzes the reinsertion of theexcised repressor gene to a random location in the genome such that therepressor gene segregates independently from the first repressiblelethal gene.

The transposase enzyme can be transiently introduced into the plantcell, or be placed under the control of an inducible promoter such thatinduction of transposition can occur. Alternatively, the transposase maybe introduced by simple sexual crossing with an isogenic or nearisogenic plant line which has been modified to express activetransposase.

Alternatively, for those embodiments which employ the use of tworepressed lethal genes and two independent repressors, the followingmethod is provided as follows:

A DNA construct is modified to contain, in addition to a firstrepressible lethal gene linked to a second repressor, a secondrepressible lethal gene linked to a first repressor. The secondrepressible lethal gene and first repressor are linked together andbounded at the 3′ and 5′ end by specific DNA sequences recognizable by atransposase enzyme. The recognition sequences are oriented in such afashion as to permit the excision of the linked second repressiblelethal gene-first repressor gene sequence by a transposase enzyme. Thetransposase further catalyzes the reinsertion of the excised genesequence to a random location in the genome such that the linked secondrepressible lethal gene—first repressor gene sequence segregatesindependently from the linked first repressible lethal gene—secondrepressor gene sequence.

The transposase activity can be provided by any means includingtransient expression of introduced DNA, direct injection of thetransposase enzyme, or more preferably, by simple sexual crossing withan isogenic or near isogenic plant line which has been modified toexpress active transposase.

Simple crossing and selection allow the selection of plant lines thatcontain both repressed lethal genes but do not contain the transposaseenzyme. Linkage of the transposase enzyme to an easily identifiablemarker gene can facilitate selection of the desired geneticcombinations. A desired combination comprises the repressed lethal geneand the repressor gene on opposite sister chromosomes of a chromosomepair.

It is within the scope of the invention to use site specificrecombination sequences to obtain site-specific insertion of repressorand repressed lethal genes. Many site specific recombinases have beendescribed in the literature (Kilby et al., Trends in Genetics, 9(12):413-418, 1993). Three recombinase systems that have been extensivelyemployed: an activity identified as R encoded by the pSR1 plasmid ofZygosaccharomyes rouxii, FLP recombinase encoded by the 2 μm circularplasmid from Saccharomyces cerevisiae and Cre-lox from the phage P1. Allof these recombinase systems have been shown to function in heterologoushosts. For example R has been demonstrated to work in tobacco cells(Onouchi et al., Nucl. Acids. Res. 19(23):6373-6378, 1991). FLP has beenshown to be functional in tobacco and Arabidopsis (Kilby et al., ThePlant Jour. 8(5):637-652, 1995), and Cre-lox has been shown to befunctional in tobacco (Russell et al., Mol. Gen. Genet. 234:49-59, 1992,Odell et al., Mol. Gen. Genet. 223:369-378, 1990, Dale and Ow, Gene91:79-85, 1990, Dale and Ow, Proc. Natl. Acad. Sci. USA 88:10558-10562,1991, Haaren and Ow, Plant Molecular Biology 23:525-533, 1993). It iswithin the scope of the present invention to target introduced DNA to aspecifically defined locus using the integration function ofsite-specific recombinases.

The use of site specific recombinases for directing homologousrecombination in higher cells is well documented. For example, Fukushigeand Sauer (Proc. Natl. Acad. Sci. USA, 89:7905-7909, 1992) demonstratedthat the Cre-lox homologous recombination system could be successfullyemployed to introduce DNA into a predefined locus in mammalian cells. Inthis demonstration a promoter-less antibiotic resistance gene modifiedto include a lox sequence at the 5′ end of the coding region wasintroduced into Chinese hamster ovary cells. Cells were re-transformedby electroporation with a plasmid that contained a promoter with a loxsequence and a transiently expressed Cre recombinase gene. Under theconditions employed, the expression of the Cre enzyme catalyzed thehomologous recombination between the lox site in the chromosomallylocated promoter-less antibiotic resistance gene and the lox site in theintroduced promoter sequence leading to the formation of a functionalantibiotic resistance gene. The authors demonstrated efficient andcorrect targeting of the introduced sequence; 54 of 56 lines analyzedcorresponded to the predicted single copy insertion of the DNA due toCre catalyzed site specific homologous recombination between the loxsequences.

Use of the Cre-lox system to specifically excise, delete or insert DNAhas been demonstrated in plants (Dale and Ow, Gene 91:79-85, 1995). Theprecise event is controlled by the orientation of lox DNA sequences. Incis, the lox sequences direct the Cre recombinase to either delete (loxsequences in direct orientation) or invert (lox sequences in invertedorientation) DNA flanked by said sequences, while in trans the loxsequences can direct a homologous recombination event resulting in theinsertion of a recombinant DNA. Accordingly, within the presentinvention a lox sequence may be first introduced into the genome of aplant cell and regenerated to a whole plant. The lox sequence serves asan “anchor” or a recombinase target DNA sequence to permit thesubsequent introduction of a recombinant DNA construct comprising arepressed lethal gene or a repressor gene or a combination thereof. Saidlox sequence may be optimally modified to further comprise a selectablemarker which is inactive but which can be activated by insertion of asequence into the lox site. It is within the scope of the presentinvention to insert into a plant cell a promoterless marker gene linkedto the lox sequence. The DNA which is to be subsequently inserted intothe target lox site is modified to contain a promoter containing a loxsite such that insertion of the DNA results in the joining of thepromoter to the promoterless marker gene, thereby activating the markergene. The Cre recombinase gene can be introduced simultaneously with theDNA insert into the plant such that insertion of the recombinant DNAinto the target lox site by homologous recombination abolishesexpression of the Cre gene.

According to the present invention, site-specific insertion ofrecombinant DNA into plant comprises:

Inserting into the genome of a transformable plant a DNA constructcomprising a DNA sequence recognized by a site specific recombinase(i.e. a recombinase target DNA sequence), and recovering a plantcontaining said sequence. The transformed plant is then made homozygousfor the recombinase target DNA sequence by selfing and selection or byanther or microspore culture as described above.

Subsequently the homozygous plants are transformed independently with:

I.) A first DNA expression cassette that comprises, in addition to theDNA sequences required for transformation and selection in plant cells,(1) a sequence that can be recognized and used by a site specificrecombinase to insert said DNA at a specific DNA sequence, and (2) arepressible lethal gene. The expression of the repressible lethal geneis regulated by an appropriate promoter, preferably a seed specificpromoter. A gene encoding a novel protein or trait may be included aspart of said first expression cassette;. and,

II.) A second DNA expression cassette that comprises, in addition to theDNA sequences required for transformation and selection in plant cells,(1) a sequence that can be recognized and used by a site specificrecombinase to insert said DNA at a specific DNA sequence, and (2) arepressor gene that encodes a repressor molecule capable of blocking theexpression of the lethal gene activity contained in the first DNAexpression cassette. Expression of the repressor gene is regulated by apromoter functional in plant cells, preferably a promoter that functionsin all plant cells, and more preferably a promoter that functions at alevel and time sufficient to inhibit expression of the lethal gene.

After transformation, plants are recovered which have integrated thesequences defined in (I) or (II) above into their genome. A recombinasefunction is introduced into the plants either in trans or by activationof a pre-existing recombinase gene to excise the DNAs described in I andII above and re-insert them at the recombinase target DNA sequence.Plants containing re-inserted recombinant DNA are recovered. Theseplants now contain either the repressible lethal gene or the repressorgene at the same locus.

Sexual crosses are carried out between the plants containing either therepressible lethal gene or the repressor gene. From those sexualcrosses, plants are selected that contain both the repressible lethalgene and the repressor gene located at the same locus on oppositechromosomes of a homologous chromosome pair.

For some embodiments of the invention, it maybe preferable to firsttransform a plant cell with the DNA encoding a recombinase enzyme whichfurther comprises the recombinase target DNA sequence. The recombinasegene may be under the control of a constitutive or tissue specificpromoter or a promoter whose expression can be conveniently regulatedsuch that the expression of the gene and subsequent site specificintegration of the repressed lethal and repressor genes can be inducedat a specific time.

In accordance with another aspect of the subject invention, methods andcompositions are provided for a novel means of producing recombinantplants that contain in addition to the fore-mentioned first and secondDNA expression cassettes, a third DNA expression cassette that comprisesa dominant conditionally lethal gene to allow plants containing theconditionally lethal gene to be killed by exposure to a chemical agent.Preferably the conditionally lethal gene is linked to the first DNAexpression cassette.

The DNA construct comprising said linked first and third DNA cassettesmay further comprise a target gene encoding a novel protein or trait.Such a novel trait cannot be transferred outside of the genotype intowhich it was introduced by crossing with native or cultivated sexuallycompatible plants. As these plants lack the repressor, any seedresulting from the union would be inviable. Plants containing the noveltrait can also be discriminated by the use of the conditionally lethalmarker gene. Accordingly, even in plant populations which may haveinadvertently received the repressor gene, the conditionally lethal genemay be used to eliminate plants which may comprise both repressor andrepressed lethal gene.

It is further noted that the conditionally lethal gene may comprise aproduct which can act both in cooperation with said repressed lethalgene to express the lethal phenotype or in response to exogenouslyapplied substances that can be acted upon to cause the expression of thelethal phenotype.

In the foregoing embodiments, segregation is used to limit thepersistence or spread of a novel trait or germplasm to unintendedpopulations.

In the most elemental form of the present invention, segregation of therepressible lethal gene, linked to a trait of interest, from therepressor gene blocks the formation of viable seed comprising the traitof interest in unintended populations through cross pollination. Undertypical agricultural conditions, if the trait of interest is linked to arepressible lethal gene, under the control of a seed-specific promoter,then persistence of the trait in an unintended plant or plantpopulation, is rapidly diluted by 75% per generation in plants capableof selfing. In this predictive model, by the F5 generation of unintendedplant population that has been cross pollinated by a plant comprising arepressible lethal gene and a repressor, the persistence of the trait ofinterest linked to the repressible lethal gene in an unintendedpopulation approaches zero. Two simple genetic models are presented, onefor plants that are primarily self pollinating, e.g., Brassica napus,and plants that are primarily self-incompatable, e.g. Brassica rapa.

Simple Genetic Model for the System in a Selfing Plant

SL = Repressible Lethal Gene under the control of a seed- specificpromoter linked to a gene of interest. R = Repressor HomozygousRepressible Seed Lethal, SL/SL, R/R Repressor Plant genotype: Crossedwith an untransformed wild plant: −/−, −/− Results in a hemizygous wildplant population comprising hemizygous lines. SL/−, R/−

If this wild plant population is a selfing plant population thefollowing progeny result:

Theoretical progeny analysis off a selfing hemizygous plant Haploidgametes SL, R SL, − −, − −, R SL, R SL/SL, R/R¹ SL/SL, −/R² SL/−, R/−³SL/−, R/R⁴ SL, − SL/SL, R/−⁵ SL/SL, −/−⁶ SL/−, −/−⁷ SL/−, −/R⁸ −, −SL/−, R/−⁹ SL/−, −/−¹⁰ −/−, −/−¹¹ −/−, −/¹² −, R SL/−, R/R¹³ SL/−, R/−¹⁴−/−, R/−¹⁵ −/−, R/R¹⁶ Persistence of the gene linked to the SL trait inthe population Plants: With Without ¹1/16 are SL/SL, R/R homozygous..0625 .0000 ^(6, 7, 10)3/16 carry only the seed lethal .0000 .1875trait, they are reproductive dead ends ^(4, 13)2/16 carry a homozygousrepressor, .0625 .0625 heterozygous SL, only half of the seed carriesthe SL gene ^(2, 5)2/16 carry a homozygous SL, .0625 .0625 hemizygous R,only half of the seed survives ^(11, 12, 15, 16)4/16 have no SL gene.0000 .2500 2/16 are hemizygous for SL, homozygous .0625 .0625 for R,this represents a one half loss of the SL gene Totals .2500 .7500

Therefore the loss of the trait linked to the SL gene is 75% pergeneration in a selfing population. This is based on the loss of thetrait with the expression of the SL in seed.

However, if the hemizygous plant is a predominantly outcrossing (orself-incompatible) plant, the persistence of a gene linked to the SLtrait is considerably lower. This is shown below.

Simple Genetic Model for the System in an Outcrossing Plant

Homozygous Repressible Seed lethal,

Homozygous Repressible Seed lethal, SL/SL, R/R Repressor Plant: Crossedwith an untransformed wild plant: −/−, −/− Results in a hemizygous wildplant population SL/−, R/− comprising hemizygous lines.

Theoretical progeny analysis of a selfing hemizygous plant Haploidgametes SL, R SL, − −, − −, R −, − SL/−, R/−¹ SL/−, −/−² −/−, −/−³ −/−,−/R⁴ −, − SL/−, R/−⁵ SL/−, −/−⁶ −/−, −/−⁷ −/−, −/R⁸ −, − SL/−, R/−⁹SL/−, −/−¹⁰ −/−, −/−¹¹ −/−, −/R¹² −, − SL/−, R/−¹³ SL/−, −/−¹⁴ −/−, /−¹⁵−/−, −/R¹⁶ Persistence of the gene linked to the SL trait in thepopulation Plants: With Without ^(1, 5, 9, 13)4/16 are SL/−, R/−hemizygous, .0625 .1875 only 75% of the population can be expected tocarry a SL and R combination (4/16 × .75) ^(2, 6, 10, 14)4/16 carry onlythe seed .0000 .2500 lethal trait, they are reproductive dead ends^(3, 7, 11, 15)4/16 have no SL gene .0000 .2500 4/16 are hemizygous forR, no SL gene .0000 .2500 Totals .0625 .9375

Therefore a plant population that is predominant outcrossing willrapidly lose a gene linked to the SL trait at a rate of 93.73% pergeneration.

It is clear from these models that the SL trait confers a selectivedisadvantage for maintenance of a gene encoding a trait of interestlinked to the repressible lethal gene in an unmanaged population (i.e.populations where the combination of repressible lethal gene andrepressor are not maintained). It should be noted that unmanagedpopulations can include sexually compatible wild species as well assexually compatible cultivated species.

In one embodiment, the repressible lethal gene is expressed by a seedspecific promoter. This allows for sexual crossing of independentlytransformed repressible lethal and repressor lines. By using therepressible lethal line as a female parent, only seed derived fromintroduction of the repressor gene will be formed, confirming thecomplete repression of the seed lethal trait. In those seeds whererepression is incomplete and hence commercially of limited value seedabortion will occur. Accordingly, this embodiment of the method providesa convenient means to select via conventional crossing the most usefulgenetic compositions.

A seed specific promoter also confers certain advantages over aconstitutive promoter for the regulation of the lethal trait. First,plants that contain the seed lethal trait can be easily converted tohomozygous lines after the introduction of the repressor gene, asfollows. A plant grown from a seed formed by the sexual introduction (orsimultaneous introduction) of the repressor and repressible seed lethalgene can be subjected to anther or isolated microspore culture todirectly recover a homozygous plant line. Alternatively, conventionalmethods such as crossing and selection of homozygous lines may be usedto recover the appropriate plant lines.

In some instances the final product or plant line for commercialpurposes may be the hemizygous combination of the repressible lethalgene and the repressor gene. According to one aspect of the invention, amethod is provided for improving hybrid seed production inself-incompatible crops using self-incompatibility. Current methods ofhybrid seed production often employ two self-incompatible lines, one ofwhich acts as a “female” parent while the other functions as a “male”parent. Under ideal conditions seed formed on the female parentrepresents hybrid seed formed as a result of pollination by the maleparent which is also self-incompatible but compatible with the femaleparent. However, such a system is prone to contamination due to theinadvertent breakdown of self-incompatibility resulting inself-pollination on the female parent. Accordingly, use of a repressiblelethal gene under the control of a seed specific promoter in the femaleparent blocks the formation of selfed seed since the seed lethal traitis expressed in selfed seed. Providing the repressor gene via the pollenof the male parent line allows formation of viable hybrid seed thatcarries a repressible lethal gene and a repressor gene.

The female parent can be increased by clonal propagation. Alternatively,since certain physical or chemical treatments can overcomeself-incompatibility, use of an inducible repressor gene repronsive tothese conditions would be advantageous. Although some of theseconditions may occur naturally in the field and would be of limitedvalue for practicing the invention, some conditions such as salt stressor high levels of carbon dioxide which are known to overcomeself-incompatibility in Brassica species, could be employed.Alternatively, a repressor active only under certain conditions (e.g. arepressor that binds to a DNA sequence in the presence of a particularsubstance) could be utilized in increasing the seed of the femaleparent. Many such repressors may be found in the art. As described, avariety of means may be employed to increase the female parent whendesired it is noted that final product of a hybrid cross is seed thatcarries a repressible lethal gene from the female parent and a repressorgene from the male parent. Said seed can further comprise a novel traitlinked to said repressible lethal gene.

In the context of the present invention any mechanism that effectivelyblocks accumulation of the product of the lethal gene in a cellcomprises repression. Such a mechanism may include the binding of aspecific “repressor protein or factor” to a DNA region or “operator”within the promoter of said lethal gene. Examples in the art include butare not limited to bacterial repressors and associated DNA bindingregions (operator DNA) such as the Lac Z repressor, the tet repressor,the class of repressor proteins that regulate sugar catabolism inbacterial systems (van Rooijen, R. J. and de Vos, W. M., J. Biol. Chem.265:18499-18503, 1990), including LacR, GutR, DeoR, FucR and GlpR, orthe Agrobacterium repressor known as accR that regulates thebiosynthesis of agrocinopines and conjugal transfer (Bodman et al.,Proc. Natl. Acad Sci USA 89:643-647, 1992). Other sources of repressorscan be employed including those found in fungi such as yeast or anyother organism. According to the present invention, the repressor iscapable of binding a specific DNA sequence present in a region of aplant promoter, said binding capable of substantially inhibitingexpression of a DNA sequence under the control of said modifiedpromoter.

It is understood that optimal expression of heterologous genes' in plantcells may require certain modifications. Typically these include:alteration of the coding sequence to reflect the usual plant codonpreferences; elimination of sequences that may be poorly recognized byplant-transcriptional or translational machinery; addition oftranslational enhancers or stabilizing sequences; and addition of DNAsequences encoding localization signals such that the protein encoded bysaid gene is correctly compartmentalized. Accordingly, for somerepressors of bacterial origin, these modifications may be required inorder to achieve sufficient expression levels of the repressor to allowfor complete repression of the repressible gene.

Other DNA binding proteins that have been modified to bind strongly tospecific DNA sequences, the so-called “transdominators”, may also beemployed as repressors. Other repressors may include antisense RNAdirected to the lethal gene, or specific inhibitors of the product ofthe lethal gene. An example is “Barstar”, a specific inhibitor of theribonuclease Barnase which is toxic when expressed in plant cells.

It is contemplated that down-regulation of the lethal gene can beaccomplished in some instances by the use of co-suppression, as long assegregation of the transgene responsible for co-suppression of thelethal phenotype restores the lethal phenotype. Accordingly repressionin the context of this invention comprises any mechanism whichreversibly inhibits the expression of the lethal phenotype.

The DNA encoding said repressible lethal gene additionally comprises apromoter region regulating the expression of said lethal gene. Althoughthe preferred embodiment comprises a seed specific promoter, otherpromoters are contemplated. Said promoter may be a constitutivepromoter, an inducible promoter, or a tissue specific promoter and maycomprise a operator sequence (repressor binding sequence) for binding aspecific repressor protein such that in the presence of said repressorproteins transcription of said lethal gene is blocked. The choice ofpromoter will be apparent to those skilled in the art and will be apromoter that in particular is known to be expressed in the plantspecies in which the invention is to be employed.

In accordance with still another aspect of the subject invention,methods and compositions are provided for a novel means of producingrecombinant plants that contain a conditionally lethal gene such thatplants containing said gene and recombinant DNA molecules can be killedby exposure to a chemical agent. The chemical agent has no effect onother plants. This mechanism completely eliminates spread of therecombinant DNA to other cultivars of the same species and relatedspecies via pollen mediated out-crossing. Conditionally lethal geneshave been described in the art and those that act directly upon anon-toxic substance to convert said substance into a toxic substance arecontemplated within the scope of the present invention.

It is further understood that a conditionally lethal gene may alsosimply comprise a repressible lethal gene capable of de-repression by aexogenously applied substance or a artificial or naturally inducedphysiological stress. Accordingly, in one specific embodiment, aconditionally lethal gene comprises a lethal gene activity which isrepressed by the binding of a DNA binding protein. Repression can belifted by a specific substance that abolishes the binding. An exampleincludes the bacterial tet repressor, whose binding to the operatorsequence is blocked by tetracycline. In this case, segregationde-represses the lethal gene activity during outcrossing orintrogression, while the gene can be further utilized as a conditionallethal gene to eliminate plants containing the recombinant DNAconstructs by exposure of the plants to tetracycline.

It is further understood that de-repression of the lethal gene activitycan also be carried out by inhibition of the expression of the repressorgene. For example, antisense RNA or ribozymes capable of inhibiting theexpression of the repressor gene can be employed. It is preferable tohave such an “anti-repressor” gene under the control of an induciblepromoter. Examples of inducible promoters that may be employed withinthe scope of the present invention include those inducible by a simplechemical such as the promoter of the 27 kD subunit of the maizeglutathione-S-transferase (GST II) gene (PCT/GB90/00110) or PR promoterssuch as PR-1a, PR-1b, PR-1c, PR-1, PR-Q, PR-S or the cucumber chitinasegene promoter, or the acidic and basic tobacco β-1,3 glucanasepromoters. Numerous chemicals capable of inducing these and relatedpromoters are described in EP89/103888.7.

Limiting the pollen-mediated movement of a target gene encoding a noveltrait involves linkage of the target gene to a repressible lethal genethat is segregated away from the repressor gene in pollen after meiosis.Activation of the repressible lethal phenotype occurs after segregationof the lethal gene from the recombinant DNA encoding the controlling orrepressing element. Accordingly, elimination of the transfer of thetarget gene to unintended sexually compatible plants is achieved. Theuse of a seed specific promoter to control the expression of therepressed lethal gene leads to non-viable seeds which results fromcross-pollination with pollen that carries the trait gene and lethalgene that expresses in the seed in the absence of the repressor. Use ofa seed specific promoter to limit the expression of the repressiblelethal gene also permits the production of pollen which ensures seed seton the recombinant plant.

Limiting the pollen-mediated movement of all recombinant DNA moleculesincluding both the trait gene linked to the repressible lethal gene andthe independently segregating repressor gene involves inclusion of asecond repressible lethal gene as a component of the first repressorgene. In this scheme, both recombinant DNAs that segregate independentlyduring meiosis carry a lethal gene; however, each lethal gene isrepressed by a distinct repressor. Accordingly, the seed-specific lethalphenotype linked to the target gene is repressed by the independentlysegregating corresponding repressor gene while expression of the secondrepressible lethal gene linked to said independently segregatingrepressor gene is repressed by a second repressor gene now linked to thetrait gene. The plant therefore carries two repressible lethal genes,each under the control of a functionally different repressor.Accordingly, one or the other or both lethal genes are derepressedfollowing meiosis and outcrossing. As a result, seed cells formed byoutcrossing or introgression of alien germplasm are inviable.

Expression of the trait of commercial interest introduced bytransformation may be regulated by a constitutive, inducible ordevelopmentally regulated promoter that may be the same or differentfrom the promoter regulating the lethal or conditionally lethalphenotype. The choice of promoter will vary in relation to the givencommercial application.

For specific aspects of the present invention where the trait ofcommercial interest is the production of heterologous proteins that areto be isolated from plant tissues, a developmentally regulated promoterfunctional in developing seeds is a logical choice. Many different typesof cell, tissue and developmentally regulated promoters are described inthe literature from which those appropriate to the trait of commercialinterest may be selected. Additionally, methods to discover andcharacterize new promoters that may be used in specific embodiments ofthe present invention are well known.

DNA encoding the novel trait can be a gene which gives rise to adetectable phenotype such as modified oil, meal, starch or other seedcomponent. Alternatively, it may be a gene which confers a particularagronomic trait such as herbicide tolerance or insect or pestresistance. The gene may also encode a protein that imparts nodetectable phenotype or a protein with pharmaceutical or industriallyuseful activity. The DNA encoding the novel trait can be expressed underthe control of a number of different promoters, depending on the trait.It is obvious to the skilled artisan that a number of strategies can beemployed for the expression of a novel trait.

For preferred embodiments of the present invention wherein therecombinant target protein of commercial interest is to be produced inand recovered from plant seeds, the first expression cassettes includesa recombinant DNA sequence comprising a transcriptional andtranslational regulatory region specifically capable of expression indeveloping plant seeds, and more specifically seed embryo or other seedtissue capable of triglyceride storage, and a second recombinant DNAsequence encoding a chimeric peptide or protein comprising a sufficientportion of an oil-body specific protein to provide targeting to an oilbody, the target protein of commercial interest and a transcriptionaland translational termination region functional in plants. The chimericpeptide or protein may also comprise a peptide sequence linking theoil-body specific portion and the target protein of commercial interestthat can be specifically cleaved by chemical or enzymatic means.

DNA expression cassettes may be so constructed that the DNA sequencescomprising the transcriptional and translational regulatory regions andthe DNA encoding both the target, repressor and lethal genes be linkedby multiple cloning sites to allow for the convenient substitution ofalternative target, repressor and lethal DNA sequences.

As preferred embodiments of the subject invention, the repressiblelethal gene activity is the oncogenes 1 and 2 from the Ti or R1 plasmidof Agrobacterium. The activity of these two genes combined leads to theproduction of IAA and plant cell death.

Oncogene 1 encodes the enzyme Indole Acetamide Synthase (IAMS) thatconverts tryptophan, an amino acid normally found in plant cells toindole acetamide. The function of oncogene 1, that is the conversion oftryptophan (a endogenous amino acid contained within all plant cells) toindole acetamide is described by VanOnckelen et al., FEBS lett. 198,357-360, 1986.

Oncogene 2 encodes the enzyme Indole Acetamide Hydrolase (IAMH) whichconverts indole acetamide to indole acetic acid. The function of gene 2,that is the ability to convert indole acetamide to indole acetic acid,was demonstrated by Tomashow et al., Proc. Natl. Acad. Sci. USA 81,5071-5075, 1984 and Schroder et al., Eur. J. Biochem. 138, 387-391,1984. Specifically oncogene 2 in concert with oncogene 1 provide for thesynthesis of the plant growth regulator indole acetic acid fromtryptophan via a pathway found in bacterial cells but not in plantcells. Related oncogene activities are found in A. rhizogenes, A. vitis(Canaday, J. et al., Mol. Gen. Genet. 235:292-303, 1992) and Pseudomonassavastanoi (Yamada et al., Proc. Natl. Acad. Sci. USA,82:6522-6526,1985).

The preferred use of oncogenes is based on the known fact that they arenaturally occurring activities that overproduce a substance normallyfound in plant cells and, that unlike lethal activities associated withtoxins such as diptheria toxin A chain, ribonucleases such as Barnaseand ribosome inhibiting proteins such as ricin and related toxins, therepression of the genetic activity need not be absolute. It has beensuggested that exceeding low levels of expression, even one molecule percell of powerful cytotoxic agents such as ricin can lead to cell death.It is noted that in order to use such powerful toxins within the scopeof this invention, repression of the repressed lethal phenotype needs tobe complete and methods are employed to achieve that level of repressionby functional assay.

Although complete repression can be easily achieved within the scope ofthis invention, such as the use of DNA binding proteins or repressors,or specific inhibitors of toxin activity (Barnase and Barstar forexample) the use of a lethal gene activity that over-expresses a growthregulator offers the opportunity to utilize a number of differentrepression schemes within the scope of the invention. Included areantisense RNA or ribozyme inhibition of the expression of the lethalgenes, preferably targeted to gene 1 or gene 1 and gene 2;co-suppression, preferably using a gene encoding a homologous sequenceto gene 1; or expression of an enzyme capable of metabolizing orconjugating excess IAA. Such enzymatic activities are known in the art.

The substrate for gene 2, indole acetamide, is not normally produced byplant cells. In addition to the conversion of indole acetamide, gene 2is capable of the metabolism of other indole amides including thesynthetic chemical naphthalene acetamide resulting in the formation ofthe powerful auxin analog naphthalene acetic acid (NAA). Application ofNAM (naphthalene acetamide) to plant cells expressing the gene 2 productIAMH produce lethal concentrations of NAA. Accordingly oncogene 2 canfunction as a conditionally lethal gene. However, within the scope ofthe present invention, oncogenes 1 and 2 preferentially comprise thelethal gene activity.

The use of both IAMS in combination with IAMH has been described as ameans to selectively ablate pollen in methods of hybrid seed production(U.S. Pat. No. 5,426,041). The possibility of using recombinantnon-native oncogene 2 alone as a conditionally lethal gene linked, in arandom fashion, to a nuclear encoded male sterility, has been suggestedas was the use of the same recombinant oncogene 2 to eliminate unwantedtransgenic plants (U.S. Pat. No. 5,180,873). However, the use of theoncogene 2 alone, without the activity of oncogene 1, fails to cause alethal gene activity. Additionally the use of the oncogene 2 as a methodto remove transgenic plants requires the application of a chemical agentin order to selectively eliminate cells containing the recombinant DNA.

By contrast, the present invention provides the inherent elimination ofplants which have inadvertently received foreign DNA without the needfor intervention. The method further employs the overexpression of acompound naturally found in plant cells to impart the lethal phenotypewhich ensures environmental safety. Accordingly the invention alsoprovides a conditionally lethal phenotype when the oncogenes 1 and 2 areused to practice the invention. In particular the unmodified, nativeoncogene 2 is employed.

In addition to oncogene 1 and 2, the use of oncogene 4 is contemplated.Oncogene 4 of the Ti plasmid of Agrobacterium sp. encodes the enzymeisopentyl transferase capable of synthesizing cytokinin, another naturalplant growth regulator. Overexpression of cytokinin can lead to celldeath and hence is a lethal gene activity within the scope of thisinvention. The growth of crown gall tumors on plants following infectionby Agrobacterium sp. is thought to result from the overexpression ofboth cytokinins and auxins due to the combined activities of oncogenes 1and 2, and oncogene 4.

It is within the scope of the present invention that the activity ofboth oncogenes 1 & 2 and oncogene 4 be repressed and employed. Thiswould require repression of both lethal gene activities. De-repressionwould result in the overexpression of growth regulators leading todestruction of the normal activity of the plant cell and hence blockingthe ability to produce seed or to reproduce. Accordingly, traits orgermplasm linked to the oncogene(s) fail to persist in the de-repressedstate.

It is further contemplated that repression of oncogenes 1, 2 and 4 canbe accomplished without the need to modify one of all of the nativegenes. For those applications which do not employ the use of a seedspecific repressible lethal gene, the native promoter of the oncogenesmay be employed and combined with a repressor molecule such as antisenseRNA or ribozymes to inhibit the expression of the oncogene.

Although oncogenes may be used methods of the present invention are notlimited by them. A variety of genes which confer lethal andconditionally lethal phenotypes can be employed within the scope of theinvention, and said methods are not limited to oncogenes. Accordinglyany gene which is capable of inhibiting proper functioning and/or growthand development of a plant cell is considered to be a lethal gene.

A number of strategies to functionally repress the activity of a lethalgene have been described. However, as more strategies have beendescribed in the art, the invention is not limited by the foregoingdescribed methods of repression. It is apparent to one skilled in theart that a variety of repression strategies may be employed within thescope of the present invention.

Any method of effecting transformation of cells and recovery oftransformed plants (such as Agrobacterium mediated DNA transfer orbiolistic methods) can be used to introduce the DNA constructs withinthe scope of the present invention. The invention is not dependent onthe method of transformation. It is further noted that the introductionof the repressible lethal gene or repressor into various plant lines mayalso be practiced by inserting the recombinant DNAs concomitantly or ina stepwise fashion. Alternatively one may obtain the desired combinationor repressible lethal and repressor genes by simple sexual crossing. Inthe instance where the genetic constructs are to be transferred tosexually incompatible relatives tissue culture techniques such as widecrosses and/or embryo rescue may be employed. A variety of techniquesknown to those skilled in the art may be employed to derive thecombination of repressible lethal and repressor genes which provides thegreatest utility within the scope of the present invention.

The following examples are set forth to illustrate the method and in noway limit the scope of the invention.

EXAMPLE 1 Isolation of Oncogene 1 and 2 from Agrobacterium Ti-plasmidpTil5955

To isolate the oncogenes, the following steps were employed. Thesubclones p101 and p202, detailed in U.S. Pat. No. 5,428,147encompassing the DNA encoding oncogene 1 (p202) and oncogene 2 (p101)are used as a source of the genes. In order to isolate the genes, acombination of PCR to introduce convenient restriction sites andsubcloning of native gene fragments is employed to derive oncogenes thatcan be conveniently inserted into plant transformation vectors.

To isolate a native oncogene 2, the following approach is used. The 5′region, including the native promoter of oncogene 2 is isolated by PCRamplification of the plasmid p101 with the following primers:

G2P1 (SEQ ID NO:1) 5′ ATA GCATGCTCTAGATGTTAGAAAAGATTCGTTTTTGTG 3′ and,G2P2 (SEQ ID NO:2) 5′ ATA CCATGG CGATCAATTTTTTTGGCGC 3′.

G2P1 contains a Sph 1 site (boldface) and a Xba I site (underlined) andcorresponds to the complement of nucleotides 5808-5785 in the publishedsequence of pTi15955. G2P2 contains a Nco 1 site (boldface) andcorresponds to nucleotides 5285-5309 in the published sequence ofpTi15955. The use of G2P1 and G2P2 yields a fragment of 523 bp whichrepresents the 5′ region of the native oncogene 2, including thepromoter modified to contain a Sph 1 and Xba I site at the 5′ end of thepromoter.

To isolate the 3′ region of oncogene 2, including the native terminatorstructure, two PCR primers are used. The first primer used is:

G2P3 (SEQ ID NO:3) 5′ ATA AAGCTT GAAAATTAAGCCCCCCCCCG 3′ and, G2P4 (SEQID NO:4) 5′ ATAGGATCCGCATGCC CAGTCTAGGTCGAGGGAGGCC 3′.

G2P3 contains a Hind III site (boldface) and corresponds to thecomplement of nucleotides 3396-3371 of the published sequence of pTi15955. G2P4 contains a Sph 1 site (boldface) and a Bam H1 site(underlined) and corresponds to nucleotides 3237-3264 of the publishedsequence of pTi 15955. The use of G2P3 and G2P4 yields a fragment of 164bp which represents a portion of the 3′ end of the native oncogene 2.

The plasmid p101 is digested with Nco I and Hind III to yield a fragmentof approximately 1895 bp fragment of oncogene 2 which encompasses mostof the coding region. The 523 bp fragment of the 5′ end of the nativeoncogene 2 is digested with Nco I and ligated to the Nco I site of the1895 bp fragment and the 164 bp 3′ end of the gene is digested with HindIII and ligated to the Hind III site of the 1895 bp fragment. Thereconstructed native oncogene 2 is then digested with Sph 1 andsubcloned into the Sph 1 site of the common cloning vector pGEM-4Z(Promega, La Jolla, Calif.). This vector is called pG2. DNA sequencingwas used to verify the composition of this reconstructed DNAcorresponding to the authentic DNA sequence of the native oncogene 2.

Isolation of oncogene 1 employs a combination of PCR to introduceconvenient restriction sites and subeloning of a native gene fragment.To isolate the required fragments, the following approach is used.Convenient restriction sites at the 5′ end of the coding region areintroduced by PCR, employing the following two primers:

G1P1 (SEQ ID NO: 5) 5′ ATA ATCGATAT AGAAACGGTTGTTGTGGTT 3′ and, G1P2(SEQ ID NO: 6) 5′ ATA AGATCT CGGGGAAGCGACC 3′

G1P1 contains a Cla 1 site (boldface) and corresponds to nucleotides5755-5775 of the published sequence of pTi 15955. G1P2 contains a Bgl IIsite (boldface) and corresponds to the complement of nucleotides6028-6010 of the published sequence of pTi 15955. G1P1 and G1P2 are usedto amplify a 273 bp fragment of oncogene 1 which is modified to containa Cla 1 site at the 5′ end of the coding region.

To isolate a 3′ fragment of the coding region of oncogene 1, two primersare used to introduce convenient restrictions sites at the 3′ end of thecoding region.

G1P3 (SEQ ID NO: 7) 5′ AAT GATATC TGAACTTTATGATAAGG 3′ and, G1P4 (SEQ IDNO: 8) 5′ ATAGAGCTCATCGAT ACTAATTTCTAGTGCGGTAGTT 3′.

G1P3 contains a Eco RV site (boldface) and corresponds to nucleotides7350-7372 of the published sequence of pTi 15955. G1P4 contains a Cla 1site (boldface) and a Sac 1 site (underlined) and corresponds tonucleotides 8076-8056 of the published sequence of pTi 15955. The use ofG1P3 and G1P4 results in a 732 bp fragment representing the 3′ end ofthe coding region of oncogene 1.

In order to reconstruct a complete coding region of the oncogene 1, theplasmid p202 is digested with Bgl II and the 1697 bp fragmentencompassing the partial coding region of the oncogene 1 is isolated. Tothe 5′ end of this fragment is added the 273 bp PCR fragment, digestedwith Bgl II, resulting in a partial oncogene 1 modified to contain a Cla1 site at the 5′ end of the coding region. To reconstruct the entireoncogene 1, the 726 bp PCR fragment representing the 3′ sequences isdigested with Bam HI and Sac 1 and the resultant fragment is ligated tothe Ban HI site at the 3′ end of the 1697 bp fragment, resulting in areconstructed oncogene 1 with Cla 1 sites at the 5′ and 3′ ends of thecoding region and a Sac 1 site at the 3′ end of the coding region. Thesefragments are contained within the vector pBluescript (Promega, LaJolla, Calif.). The resulting plasmid is called pG1. DNA sequencing wasused to verify the composition of this reconstructed DNA correspondingto the authentic DNA sequence of the native oncogene 1.

A diagrammatic representation of the steps employed in the constructionof pG1 and pG2 is shown in FIGS. 7 a and 7 b.

EXAMPLE 2 Construction of a Phaseolin Promoter with a BacterialRepressor Binding Site

In this example, the tetracycline (tet) operator DNA is introduced intothe phaseolin promoter. In order to insert the tet operator sequenceinto the phaseolin promoter sequence, PCR is used to isolate the regionof the promoter that corresponds the DNA sequence 5′ to the native TATAbox and a synthetic DNA sequence containing three copies of the tetoperator sequence. The TATA box is ligated to the PCR fragment of thepromoter resulting in the formation of a reconstructed phaseolinpromoter containing three copies of the tet operator sequence. The meansby which this is accomplished is as follows and is shown in FIG. 8.

The promoter region of the phaseolin gene (described in: Slightom, J.L., Sun, S. M. and Hall, T. C., Proc. Natl. Acad. Sci. USA 80:1897-1901,1983) is isolated by PCR using the vector pAGM 219, kindly supplied byDr. G. Cardineau of Mycogen Plant Sciences, San Diego, Calif. Theplasmid pAGM 219 contains approximately 1600 base pairs of the promoterregion of the phaseolin gene and the native termination region of thephaseolin gene. The region of the promoter 5′ to the TATA box wasisolated by PCR in preparation for the addition of a synthetic DNAsequence comprising the tet operator DNA and a TATA box.

The first PCR primer used was engineered to introduce a Csp45 1 site bya minor alteration of the nucleotide sequence in the native promotersequence. The sequence of this primer is shown below:

SEQ ID NO: 9 5′GGTGG TTCGAA CATGCATGGAGATTTG 3′

The Csp45 1 restriction site is shown in boldface. The second primerused for PCR has the following sequence:

SEQ ID NO: 10 5′CCGTAT CTCGAG ACACATCTTCTAAAGTAATTT 3′

A Xho 1 site is indicated in boldface. The PCR product obtained usingthese primers was called pPHAS and corresponds to nucleotides 128 - 833of the DNA sequence of the phaseolin promoter of the lambda genomicclone AG-λPVPh177.4 (λ177.4), (Slightom, J.L., Sun, S.M. and Hall, T.C.,Proc. Nati. Acad. Sci. USA 80:1897-1901, 1983). A synthetic tet operatorsequence was added to this fragment by joining the synthetic duplex DNAto the Csp45 1 site in the PCR product. The synthetic operator DNAsequence also comprises a Cla 1 site at the 3′ end of the sequence. Thetop strand of the synthetic DNA has the following sequence:

SEQ ID NO: 11 5′ TTCGAA G ACTCTATCAGTGATAGAGT GTATATAAG ACTCTATCAGTGATAGAGT GA ACTCTATCAGTGATACAGT AT

3′

Which comprises 3 copies of the operator DNA (boldface), a TATA box(underlined), a Csp45 1 site at the 5′ end (italics and underlined) anda Cia 1 site at the 3′ end (italics and boldface). A bottom strandfragment is used which has the following sequence:

SEQ ID NO: 12 5′ CG ATATACTGTATCACTGATAGAGTTCACTCTATCACTGATAGAGTCTTATATACACTCTATCACTGATAGAGTCTTCGTT 3′.

Which comprises a complementary strand to SEQ ID NO:9 and contains a Cla1 cohesive end, identified in boldface. The duplex DNA is referred to a“top” DNA and is ligated to the Csp45 1 and Cla 1 cut pPHAS and clonescontaining the inserted “top” DNA are chosen. This vector is referred toas pPHAStet1. DNA sequencing was used to verify the composition of thisreconstructed DNA.

EXAMPLE 3 Construction of a Plant Transformation Vector Comprising anOncogene 1 Under the Control of a Modified Repressible PhaseolinPromoter Linked to an Active Oncogene 2.

In this example, formation of a plant transformation vector is describedwhich comprises a repressible lethal gene activity resulting from thecombined activity of two genes, oncogene 1 (placed under the control ofthe modified phaseolin promoter) and native oncogene 2. When expressed,the two oncogenes in this vector lead to the formation of excess IAA,killing plant cells in which the lethal gene activity is expressed. Toconstruct this vector, the following steps are employed.

The plasmids pPHAStet1 and pG1 are digested with Cla 1 and the codingregion for oncogene 1 is inserted into the Cla 1 site of pPHAStet1 toproduce the vector pPG-1. The phaseolin terminator contained in theplasmid pAGM 219, comprising the nucleotide sequences starting at 36 bpdownstream of the protein termination codon TGA comprising a Sac I siteextending approximately 1400 nucleotides ending at a Pst 1 site, wasfurther modified to introduce a Pst 1 site at the position of the Sac 1site. This modification allows the entire terminator sequence to beexcised from pAGM 219 as a Pst 1 fragment of approximately 1400 bp. This1400 bp terminator fragment is inserted into the Pst 1 site of pPG-1 toform pPG-2, which comprises the phaseolin promoter modified to containthree tet operator DNA sequences, the coding region of oncogene 1 andthe phaseolin terminator sequence.

The Sph 1 fragment of pG2 containing the native oncogene 2 is insertedinto the unique Sph 1 site of pPG-2 to form the vector pGG-1. The vectorpGG-1 is digested with Xba I to excise the entire insert comprising thephaseolin promoter modified to contain three tet operator DNA sequences,the coding region of oncogene 1 and the phaseolin terminator sequence,and the native oncogene 2 under the control of its own promoter. ThisXba I fragment is inserted into the Xba I site of the planttransformation vector Binter.

The plant transformation vector Binter comprises the widely used planttransformation vector Bin 19 (Clontech, Palo Alto, Calif.) into whichhas been inserted a nos terminator fragment as follows. The nosterminator contained in the vector pBI 221 (Clontech) was first isolatedas a Sac 1-Eco R1 fragment and cloned into pGEM-4Z at the Sac I and EcoR1 sites. This plasmid, pGEMter, was digested with Hind III and Eco R1to remove the nos terminator and the entire polylinker and inserted intoHind III-Eco R1 digested Bin 19. This vector is called Binter.

Binter containing the Xba I fragment comprising the phaseolin promotermodified to contain three tet operator DNA sequences, the coding regionof oncogene 1 and the phaseolin terminator sequence, and the nativeoncogene 2 under the control of its own promoter is referred to aspGG-2. The steps employed to construct pGG-2 are illustrated in FIG. 9.

EXAMPLE 4 Transformation of Plants to Introduce a Repressible SeedLethal Gene Under the Control of a Modified Phaseolin Promoter

In this example, tobacco plants are transformed with the vector pGG-2using standard Agrobacterium mediated transformation to obtain plantswhich comprise a repressible seed lethal gene activity. Plants obtainedwere grown in the greenhouse and allowed to flower. Selfed seed wascollected as well as seed derived from reciprocal crossing withwild-type tobacco. Tobacco plants that carry the repressible seed lethalgene but do not carry a repressor form seeds that are not viable asjudged from germination assays. This is illustrated in FIGS. 10 and 11.In FIG. 10, a photomicrograph of germinating tobacco seeds comprisingthe seed lethal vector and wild-type tobacco seeds are compared. Seedswere surface sterilized and plated on basic media. Seeds were allowed togerminate. Wild-type seeds are marked “WT” and seeds comprising the seedlethal gene are identified as “SL”. Wild-type seeds geminated normally,had normal cotyledons and true first leaves. “SL” plantlets hadthickened cotyledons, lacked true first leaves and showed typical signsof auxin overproduction, including callus and excessive rooty phenotype.In order to provide evidence that the normal plantlets or the “WT”plantlets were devoid of the seed lethal construct, seeds from the sametransformant were planted on media with and without kanamycin. Followinggermination on kanamycin containing media, all of the normal plantletsbecame bleached and died, indicating that they did not contain the seedlethal construct (that is linked to the kanamycin gene in the pGG-2transformation vector) and hence were sensitive to kanamycin. This isshown in FIG. 11. In this experiment, the plate marked “-kan” shows amixture of seed lethal and wild-type tobacco plantlets from seedgerminated on media without kanamycin. In the plate labeled “+kan”, asimilar sampling of seeds of seed lethal and wild-type were germinatedin the presence of 300 ugs per ml of kanamycin. The seed lethalphenotype is visible in both plates, the seed lethal plantlets haveexcessive roots, thickened cotyledons and lack true first leaves. In theplate without kanamycin, the wild-type seeds produce normal plantlets,on plates with kanamycin the wild-type plants can not grow and theplantlets become bleached and eventually die. All of the seed lethalplantlets remained green, even though they failed to form normalplantlets. Thus the seed lethal phenotype is dependent on the presenceof the seed lethal gene. It is also clear, that based on the results ofthe reciprocal crosses, the seed lethal phenotype can be transmitted viapollen. Thus the seed lethal phenotype is only manifested in the seed,and does not effect other tissues of the plant.

EXAMPLE 5 Introduction of a Repressible Lethal Gene and Repressor Geneinto a Plant Line

In the first portion of this example, tobacco plants that carry therepressible lethal gene under the control of a modified phaseolinpromoter are used as a female parent in a cross with tobacco that waspreviously transformed with a gene encoding the tet repressor under thecontrol of a ³⁵S promoter and is homozygous for the inserted repressorgene. Plants appear phenotypically normal (FIG. 12). In FIG. 12, plant Ais a plant that contains a repressible seed lethal gene, plant B is aplant that contains a repressor gene, plant C is a plant derived bycrossing plants A and B, while plant D is a wild-type tobacco plants. Itshould be noted that the plants are not all exactly the same age; theseplants were maintained by propagation. This photograph is provided toillustrate that plants with a repressible seed lethal gene arephenotypically normal. Seed from plant C is recovered and germinated inthe presence of kanamycin to select seed that contains the repressiblelethal gene. Viable seed (i.e. seed that germinates normally) containsboth the repressible lethal gene and a copy of the repressor gene. PCRanalysis for the presence of the repressible lethal gene and therepressor confirmed the genotype. Phenotype was scored by germinationanalysis. A significant number of independently transformed lines wereobtained and analyzed as above. Most of these independent transformedplants exhibited a seed lethal phenotype that ranged from seedscompletely unable to germinate to seeds that germinated but yieldedabnormal plantlets with excessive roots, thickened cotyledons and lacktrue first leaves. Table 1 contains the summary data from a series ofcrosses carried out with representative samples of these various plants.The plants identified were tested for the presence of the seed lethalgene (abbreviated as “SL” in column 2), scored for seed viability incolumn 3, crossed with the indicated repressor line (abbreviated as “R”in column 4), seed collected from these plants were analyzed bygermination assays (as indicated in column 5) and plant tissue analyzedfor the presence of the repressor and seed lethal gene. This analysisproved that repression of the seed lethal gene by the repressorpermitted the formation of viable seed. Viable seed germinated normallyand was found to contain both the repressible seed lethal gene and therepressor.

TABLE 1 Summary Data from Crosses of Plants Containing Repressible SeedLethal Genes and Plants Containing Repressor Genes. Column 4 ColumnColumn Crossed Column Column Column 2 3 with 5 6 1 Genotype SeedRepressor Genotype(by Seed Plant # (by PCR) Viability Plant PCR)Viability PL2 SL Gene Non-viable R17-X SL gene, R Viable seed gene seedPL3 SL Gene Non-viable R17-X SL gene, R Viable seed gene seed PL4 SLGene Non-viable R17-X SL gene, R Viable seed gene seed PL5 SL GeneNon-viable R17-X SL gene, R Viable seed gene seed PL6 SL Gene Non-viableR17-X SL gene, R Viable seed gene seed PL17 SL Gene Non-viable R17-X SLgene, R Viable seed gene seed PL21 SL Gene Non-viable R17-X SL gene, RViable seed gene seed PL38 SL Gene Non-viable R17-X SL gene, R Viableseed gene seed PL48 SL Gene Non-viable R17-X SL gene, R Viable seed geneseed PL53 SL Gene Non-viable R17-X SL gene, R Viable seed gene seed WildNo SL Viable seed R17-X R gene Viable type gene seed

Plant lines that contained a repressible seed lethal gene were crossedwith a plant line containing a repressor gene. Segregating seedpopulations of both the original plant lines containing the repressibleseed lethal gene and plant lines from those plants crossed with arepressor line were germinated in soil. It was found that within asegregating population of seeds derived from a plant containing a seedlethal gene, only those segregants that did not have the seed lethalgene grew. No plants were recovered that carried a seed lethal gene,proving that without the presence of a repressor, no viable plants canbe formed from seeds with a seed lethal genotype. However, normal plantswere recovered from the seed of crosses with a repressor. These normalplants comprised both the seed lethal and the repressor genes. Thisindicates that repression of the seed lethal phenotype can be achievedunder normal growth conditions. When the seed lethal gene and therepressor gene segregate following crosses with wild-type plants, theseed lethal phenotype re-appears, indicating the repression has beenlost through segregation. Thus these series of experiments indicates themethod works as predicted based on the genetic model.

It is clear from the foregoing examples, that derivation of geneticcombination comprising a repressible seed lethal gene and a repressor iswithin the ordinary skill of those in the art. Various modifications tothe method such as derivation of homozygous lines, different crossingprocedures or re-transformation of plant lines to combine therepressible lethal gene and the repressor are also apparent and arefully appreciated by the skilled artisan.

EXAMPLE 6 Production of a Homozygous Plant Line

The plants obtained in example 5, above, illustrate the utility of themethod and it is appreciated that a variety of similar steps may beemployed with different crops. As an illustration of how a geneticcombination of the invention is achieved in a crop such as oilseedBrassica napus, this example describes the use of the method incombination with anther culture to rapidly obtain homozygous plantlines. In the present example, the first step is obtaining a plant thatcarries a seed lethal trait by transforming a Brassica napus plant witha recombinant DNA construct comprising a repressible lethal genepreferably linked to an easily identifiable marker gene such as the GUSgene. From a population of primary transformants of plants transformedwith a single copy of a repressible lethal gene one would identify thoseplants in which the seed lethal trait is expressed; thus plants areunable to produce any seed that carries the genetic construct asidentified by GUS screening of selfed seeds. Such a plant is thensubjected to anther culture to convert the plant to a double haploidplant incapable of producing selfed seed. This plant is homozygous forthe repressible lethal gene.

In order to derive a repressor-containing plant capable of repressingthe seed lethal phenotype, a Brassica napus plant, preferably of thesame plant variety, is transformed with a recombinant DNA constructcomprising the repressor DNA. A population of plants containing saidrepressor DNA is selected. This population of repressor-containingplants is used as male parent to cross to the plant that expresses theseed lethal trait. To simplify the recovery of the crossed seed, it ispreferable to emasculate the female parent during the cross. Viable seedproduced as a result of said cross must contain the repressible lethalgene and a repressor gene capable of repressing the seed lethalphenotype. Seed from each individual cross is recovered and grown out.From this population of plants, an individual plant is selected which iscapable of full self seed set and carries a repressible lethal seedtrait. When cross-pollinated to other varieties lacking the repressorgene, such a plant is substantially unable to produce seeds which arepositive for the GUS activity linked to said repressed lethal gene.

By such method, the genetic combination can be selected that mostefficiently restricts out crossing while maintaining full self-seed setability. It is not necessary to carry out detailed mapping of theinserted DNA, since the most favorable genetic combination will be thoseplants which contain the inserted DNAs in the genetic loci that mosteffectively segregate during meiosis. The seed from this plant linerepresents the original starting variety modified only to contain arepressible lethal gene and repressor gene. However said plant line isunable to substantially transfer any trait or traits associated with therepressible lethal gene.

For the production of hybrid crops such as hybrid Brassica napus, whichcarry a repressed lethal gene and a repressor, a modification of themethod is provided to allow the production of parental lines. The mostobvious approach to combining the repressible lethal gene and therepressor gene is during the production of the hybrid seed. Accordinglyhybrid seed thus produced will carry the genetic composition of arepressible lethal gene and a repressor gene.

In order to accomplish the production of this genetic composition, ameans to increase seed of the male parent is provided. This methodincludes transformation of a plant with a DNA molecule comprising arepressible lethal gene and a DNA molecule comprising a repressor geneunder the control of an inducible promoter. Preferably the genes arelinked and may further comprise a novel trait. The induction of thepromoter allows the plant to be made homozygous for the repressed lethalgene and inducible repressor gene. Plant seeds obtained can serve as amale parent in a hybrid cross. Seed is increased in the presence of theinducer.

The female parent in said hybrid cross is produced by transforming aplant with a DNA molecule comprising a repressor gene, making the planthomozygous for the repressor gene; permitting self pollination andself-seed formation; and using this plant line as a female parent in ahybrid cross.

1. A method of producing a genetically modified plant, comprising: (a)providing at least one plant cell capable of being transformed and beinggenerated into a whole plant; (b) introducing into the at least oneplant cell: (I) a repressible lethal gene encoding a gene product havingan activity lethal to plant cells, wherein said repressible lethal geneis selected from the group consisting of: a gene encoding the proteinencoded by oncogene 1 of Agrobacterium; a gene encoding the proteinencoded by oncogene 2 of Agrobacterium; and a gene encoding the proteinencoded by oncogene 4 of Agrobacterium; and (ii) a sense repressor geneencoding a protein capable of repressing the activity of the geneproduct of the repressible lethal gene; wherein a DNA operator sequenceis in operable association with the repressible lethal gene, the DNAoperator sequence adapted for binding the gene product of the repressorgene to repress transcription of the gene product of the repressiblelethal gene; (c) generating a plurality of whole plants from the atleast one plant cell; and (d) selecting for a genetically modified plantdescended from or derived from at least one of the plurality of wholeplants by determining incorporation and mutually independent segregationof the repressor gene and the repressible lethal gene within thegenetically modified plant.
 2. The method of claim 1, wherein saidintroducing further comprises providing the repressible lethal gene in afirst vector construct and providing the repressor gene in a secondvector construct, and further comprising crossing at least two plants ofthe plurality of whole plants prior to said selecting.
 3. The method ofclaim 1, wherein said determining mutually independent segregation ofthe repressor gene and the repressible lethal gene comprises determiningthat the repressible lethal gene and the repressor gene are located onrespective opposite sister chromosomes of a chromosome pair of a plantcell of the genetically modified plant.
 4. The method of claim 1,further comprising providing a tissue-specific promoter intranscriptional control of at least one of the repressible lethal geneor the repressor gene.
 5. The method of claim 4, wherein said providingthe tissue-specific promoter comprises providing a seed-specificpromoter.
 6. The method of claim 5, wherein the seed-specific promoteris a phaseolin promoter.
 7. The method of claim 1, further comprisingproviding an inducible promoter in transcriptional control of therepressor gene.
 8. The method of claim 1, wherein the activity of thegene product of the repressible lethal gene comprises over-expression orunder-expression of a naturally occurring plant growth regulatingsubstance in a plant cell of the genetically modified plant.
 9. Themethod of claim 1, further comprising linking a gene conferring a traitof interest with the repressible lethal gene in a first vectorconstruct, and wherein said introducing comprises introducing the firstvector construct to the at least one plant cell.
 10. The method of claim1, wherein said generating the plurality of whole plants comprisesgenerating at least one plant which is homozygous for the repressiblelethal gene and the repressor gene.
 11. The method of claim 10, furthercomprising crossing the at least one plant which is homozygous for therepressible lethal gene and the repressor gene with a second plant toproduce the genetically modified plant.
 12. A method of producing agenetically modified plant having at least one repressible lethal geneexpressed during outcrossing or introgression of alien germplasm,comprising: (a) providing a plant cell capable of being transformed andbeing regenerated to a whole plant; (b) introducing into the plant cell:(I) a first repressible lethal gene under transcriptional control of aseed-specific promoter, the first repressible lethal gene encoding agene product having a first gene product activity lethal to plant cells,wherein said first repressible lethal gene is selected from the groupconsisting of: a gene encoding the protein encoded by oncogene 1 ofAgrobacterium; a gene encoding the protein encoded by oncogene 2 ofAgrobacterium; and a gene encoding the protein encoded by oncogene 4 ofAgrobacterium; (ii) a first operator sequence in operable associationwith the first repressible lethal gene; (iii) a first bacterialrepressor gene, the first bacterial repressor gene encoding a proteincapable of repressing the first gene product activity by binding to thefirst operator sequence; (iv) a second repressible lethal gene undertranscriptional control of a seed-specific promoter, the secondrepressible lethal gene encoding a gene product having a second geneproduct activity lethal to said plant cells , wherein said secondrepressible lethal gene is selected from the group consisting of: a geneencoding the protein encoded by oncogene 1 of Agrobacterium; a geneencoding the protein encoded by oncogene 2 of Agrobacterium; and a geneencoding the protein encoded by oncogene 4 of Agrobacterium; (v) asecond operator sequence in operable association with the secondrepressible lethal gene; (vi) a second repressor gene, the secondrepressor gene encoding a protein capable of repressing the second geneproduct activity by binding to the second operator sequence; (vii) atleast one gene conferring a trait of interest linked to at least one ofthe first and second repressible lethal genes; (c) regenerating a wholeplant from the plant cell; and (d) selecting for a genetically modifiedplant descended from or derived from the whole plant by determiningincorporation and mutually independent segregation of the firstrepressor gene from the first repressible lethal gene, and bydetermining incorporation and mutually independent segregation of thesecond repressor gene from the second repressible lethal gene within thegenetically modified plant.
 13. The method of claim 12, wherein saidintroducing further comprises providing the first repressible lethalgene, the at least one gene conferring the trait of interest, the firstoperator sequence and the second repressor gene in a first geneticconstruct, and providing the second repressible lethal gene, the secondoperator sequence and the first repressor gene in a second geneticconstruct.
 14. The method of clam 12, wherein said introducing comprisesintroducing the first repressible lethal gene, the first operatorsequence, the at least one gene conferring the trait of interest, thesecond repressor gene, the second repressible lethal gene, the secondoperator sequence and the first repressor gene to the plant cell in asingle transformation vector.
 15. A method of producing a geneticallymodified plant having at least one repressible lethal gene expressedduring outcrossing or introgression of alien germplasm, comprising: (a)providing a plant cell capable of being transformed and beingregenerated to a whole plant; (b) introducing into the plant cell: (I) arepressible lethal gene under transcriptional control of a seed-specificpromoter, the repressible lethal gene encoding a gene product having agene product activity lethal to plant cells, wherein said repressiblelethal gene is selected from the group consisting of: a gene encodingthe protein encoded by oncogene 1 of Agrobacterium; a gene encoding theprotein encoded by oncogene 2 of Agrobacterium; and a gene encoding theprotein encoded by oncogene 4 of Agrobacterium; (ii) an operatorsequence in operable association with the first repressible lethal gene;(iii) a bacterial repressor gene, the bacterial repressor gene encodinga protein capable of repressing the lethal gene product activity bybinding to the operator sequence; (iv) at least one gene conferring atrait of interest linked to the repressible lethal gene; (c)regenerating a whole plant from the plant cell; and (d) selecting for agenetically modified plant descended from or derived from the wholeplant by determining incorporation and mutually independent segregationof the repressor gene from the repressible lethal gene.
 16. A plant, ora descendant, cell, tissue, or part thereof, comprising: (I) arepressible lethal gene encoding a gene product having an activitylethal to plant cells, wherein said repressible lethal gene is selectedfrom the group consisting of: a gene encoding the protein encoded byoncogene 1 of Agrobacterium; a gene encoding the protein encoded byoncogene 2 of Agrobacterium; and a gene encoding the protein encoded byoncogene 4 of Agrobacterium; and (ii) a sense repressor gene encoding aprotein capable of repressing the activity of the gene product of therepressible lethal gene; said repressible lethal gene and said repressorgene being stably integrated into the genome of said plant such thatsaid repressible lethal gene segregates independently from saidrepressor gene.
 17. A plant comprising at least one plant cell derivedfrom or descended from a genetically modified plant produced by themethod of claim 12, said plant cell comprising: (I) a first repressiblelethal gene under transcriptional control of a seed-specific promoter,the first repressible lethal gene encoding a gene product having a firstgene product activity lethal to plant cells, wherein said firstrepressible lethal gene is selected from the group consisting of: a geneencoding the protein encoded by oncogene 1 , of Agrobacterium; a geneencoding the protein encoded by oncogene 2 of Agrobacterium; and a geneencoding the protein encoded by oncogene 4 of Agrobacterium; (ii) afirst operator sequence in operable association with the firstrepressible lethal gene; (iii) a first bacterial repressor gene, thefirst bacterial repressor gene encoding a protein capable of repressingthe first gene product activity by binding to the first operatorsequence; (iv) a second repressible lethal gene under transcriptionalcontrol of a seed-specific promoter, the second repressible lethal geneencoding a gene product having a second gene product activity lethal tosaid plant cells, wherein said second repressible lethal gene isselected from the group consisting of: a gene encoding the proteinencoded by oncogene 1 of Agrobacterium; a gene encoding the proteinencoded by oncogene 2 of Agrobacterium; and a gene encoding the proteinencoded by oncogene 4 of Agrobacterium; (v) a second operator sequencein operable association with the second repressible lethal gene; (vi) asecond repressor gene, the second repressor gene encoding a proteincapable of repressing the second gene product activity by binding to thesecond operator sequence; and (vii) at least one gene conferring a traitof interest linked to at least one of the first and second repressiblelethal genes; said first and second repressible lethal genes and saidfirst and second repressor genes being stably integrated into the genomeof said plant such that said first repressible lethal gene segregatesindependently from said first repressor gene and said second repressiblelethal gene segregates independently from said second repressor gene.18. A plant comprising at least one plant cell derived from or descendedfrom a genetically modified plant produced by the method of claim 15,said plant cell comprising: (I) a repressible lethal gene undertranscriptional control of a seed-specific promoter, the repressiblelethal gene encoding a gene product having a gene product activitylethal to plant cells, wherein said repressible lethal gene is selectedfrom the group consisting of: a gene encoding the protein encoded byoncogene 1 of Agrobacterium; a gene encoding the protein encoded byoncogene 2 of Agrobacterium; and a gene encoding the protein encoded byoncogene 4 of Agrobacterium; (ii) an operator sequence in operableassociation with the repressible lethal gene; (iii) a bacterialrepressor gene, the bacterial repressor gene encoding a protein capableof repressing the lethal gene product activity by binding to theoperator sequence; and (iv) at least one gene conferring a trait ofinterest linked to the repressible lethal gene; said repressible lethalgene and said repressor gene being stably integrated into the genome ofsaid plant such that said repressible lethal gene segregatesindependently from said repressor gene.
 19. A plant, or a descendant,cell, tissue, or part thereof, comprising: (a) a repressible lethal geneencoding a gene product having an activity lethal to plant cells,wherein said repressible lethal gene is selected from the groupconsisting of: a gene encoding the protein encoded by oncogene 1 ofAgrobacterium; a gene encoding the protein encoded by oncogene 2 ofAgrobacterium; and a gene encoding the protein encoded by oncogene 4 ofAgrobacterium; (b) an operator sequence in operable association withsaid repressible lethal gene; and (c) a bacterial repressor geneencoding a protein capable of repressing said repressible lethal gene bybinding to said operator sequence; said repressible lethal gene and saidrepressor gene being stably integrated into the genome of said plantsuch that said repressible lethal gene segregates independently fromsaid repressor gene.
 20. The plant or descendant, cell, tissue, or partthereof according to claim 19, wherein said repressible lethal gene andsaid repressor gene are located on respective opposite sisterchromosomes of a chromosome pair of said plant.
 21. The plant ordescendant, cell, tissue, or part thereof according to claim 19, whereinsaid repressible lethal gene comprises a gene encoding the proteinencoded by oncogene 1 of Agrobacterium and a gene encoding the proteinencoded by oncogene 2 of Agrobacterium.
 22. The plant or descendant,cell, tissue, or part thereof according to claim 19, wherein saidoperator sequence is a tetracycline (tet) operator sequence, and saidbacterial repressor protein is a protein capable of binding said tetoperator sequence.
 23. The plant or descendant, cell, tissue, or partthereof according to claim 19, further comprising at least one geneconferring a trait of interest linked to said repressible lethal gene.24. The plant or descendant, cell, tissue, or part thereof according toclaim 19, wherein at least one of said repressible lethal gene and saidrepressor gene is under transcriptional control of a tissue-specificpromoter.
 25. The plant or descendant, cell, tissue, or part thereofaccording to claim 24, wherein said tissue-specific promoter is aseed-specific promoter.
 26. The plant or descendant, cell, tissue, orpart thereof according to claim 25, wherein said seed-specific promoteris a phaseolin promoter.
 27. The plant or descendant, cell, tissue, orpart thereof according to claim 19, which is a Brassica plant ordescendant, cell, tissue, or part thereof.
 28. The plant or descendant,cell, tissue, or part thereof according to claim 19, which is a Brassicanapus plant or descendant, cell, tissue, or part thereof.
 29. A Brassicanapus plant, or a descendant, cell, tissue, or part thereof, comprising:(a) a repressible lethal gene encoding a gene product having an activitylethal to plant cells said repressible lethal gene comprising anoncogene; and (b) a repressor gene encoding a gene product capable ofrepressing the activity of the gene product of said repressible lethalgene; said repressible lethal gene and said repressor gene being stablyintegrated into the genome of said plant such that said repressiblelethal gene segregates independently from said repressor gene; whereinsaid repressible lethal gene comprises a gene encoding the proteinencoded by oncogene 1 of Agrobacterium and a gene encoding the proteinencoded by oncogene 2 of Agrobacterium; and wherein said gene encodingthe protein encoded by oncogene 1 of Agrobacterium is undertranscriptional control of a phaseolin promoter modified to comprise atetracycline (tet) operator sequence, and said repressor protein is abacterial protein capable of binding said tet operator sequence.
 30. Theplant or descendant, cell, tissue, or part thereof according to claim16, wherein said repressible lethal gene comprises a gene encoding theprotein encoded by oncogene 1 of Agrobacterium and a gene encoding theprotein encoded by oncogene 2 of Agrobacterium.
 31. The plant ordescendant, cell, tissue, or part thereof according to claim 16, furthercomprising at least one gene conferring a trait of interest linked tosaid repressible lethal gene.
 32. The plant or descendant, cell, tissue,or part thereof according to claim 16, wherein at least one of saidrepressible lethal gene and said repressor gene is under transcriptionalcontrol of a tissue-specific promoter.
 33. The plant or descendant,cell, tissue, or part thereof according to claim 32, wherein saidtissue-specific promoter is a seed-specific promoter.
 34. The plant ordescendant, cell, tissue, or part thereof according to claim 33, whereinsaid seed-specific promoter is a phaseolin promoter.
 35. The plant ordescendant, cell, tissue, or part thereof according to claim 16, whichis a Brassica plant or descendant, cell, tissue, or part thereof. 36.The plant or descendant, cell, tissue, or part thereof according toclaim 16, which is a Brassica napus plant or descendant, cell, tissue,or part thereof.